Antibodies that immunospecifically bind to trail receptors

[0001] This application claims the benefit of priority under 35 U.S.C. 119(e) based on U.S. Provisional Application Serial Nos. 60/341,237 filed Dec. 20, 2001; 60/369,877 filed Apr. 05, 2002; 60/384,828 filed Jun. 04, 2002; 60/396,591 filed Jul. 18, 2002; 60/403,370 filed Aug. 15, 2002; and 60/425,737 filed Nov. 13, 2002. Each of the above U.S. Provisional Patent applications is hereby incorporated by reference in its entirety.

[0002] The present invention relates to antibodies and related molecules that immunospecifically bind to TRAIL receptor, TR7. Such antibodies have uses, for example, in the prevention and treatment of cancers and other proliferative disorders. The invention also relates to nucleic acid molecules encoding anti-TR7 antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder, especially cancer and other hyperproliferative disorders, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR7.

[0003] Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intra-cellular response.

[0004] For example, tumor necrosis factors (TNF) alpha and beta are cytokines which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease. The TNF molecules belong to the TNF-ligand superfamily, and act together with their receptors or counter-ligands, the TNF-receptor superfamily. So far, at least eighteen members of the TNF ligand superfamily have been identified and at least nineteen members of the TNF-receptor superfamily have been characterized (See, e.g., Locksley et el., Cell (2001) 104:487-501).

[0005] Among the ligands there are included TNF-, lymphotoxin- (LT-, also known as TNF-), LT- (found in complex heterotrimer LT-(2-), FasL, CD40L, CD27L, CD30L, 4-lBBL, OX40L and nerve growth factor (NGF). The superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-lBB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).

[0006] Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).

[0007] Considerable insight into the essential functions of several members of the TNF receptor family has been gained from the identification and creation of mutants that abolish the expression of these proteins. For example, naturally occurring mutations in the FAS antigen and its ligand cause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature 356:314 (1992)), perhaps reflecting a failure of programmed cell death. Mutations of the CD40 ligand cause an X-linked immunodeficiency state characterized by high levels of immunoglobulin M and low levels of immunoglobulin G in plasma, indicating faulty T-cell-dependent B-cell activation (Allen, R. C. et al., Science 259:990 (1993)). Targeted mutations of the low affinity nerve growth factor receptor cause a disorder characterized by faulty sensory innovation of peripheral structures (Lee, K. F. et al., Cell 69:737 (1992)).

[0008] TNF and LT- are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors). A large number of biological effects elicited by TNF and LT-, acting through their receptors, include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation, proliferation and anti-viral responses, as well as protection against the deleterious effects of ionizing radiation. TNF and LT- are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection.

[0009] Moreover, an about 80 amino acid domain near the C-terminus of TNFR1 (p55) and Fas was reported as the death domain, which is responsible for transducing signals for programmed cell death (Tartaglia et al., Cell 74:845 (1993)).

[0010] Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H. Steller, Science 267, 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C. B. Thompson, Science 267, 1456-1462 (1995)). Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland, et al., Cell 81, 479-482 (1995); A. Fraser, et al., Cell 85, 781-784 (1996); S. Nagata, et al., Science 267, 1449-56 (1995)). Both are members of the TNF receptor family which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (C. A. Smith, et al., Science 248, 1019-23 (1990); M. Tewari, et al., in Modular Texts in Molecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the death domain, which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185-6 (1995); K. White et al., Science 264, 677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Activation of Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270, 7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588 (1995)), which in turn binds and presumably activates FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85, 817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996)). While the central role of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today 13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu, et al., Cell 81, 495-504 (1995); H. Hsu, et al., Cell 84, 299-308 (1996)). Through its associations with a number of signaling molecules including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, et al., Immunity 4, 387-396 (1996)).

[0011] One TNF-related apoptosis inducing ligand has been reported by several groups and has been ascribed the name Apoptosis Inducing Molecule I (AIM-I) (Intenation Application No. WO 97/33899) and TNF-related apoptosis-inducing ligand or (TRAIL) (Wiley, S. R. et al., Immunity 3:673-682 (1995)). Pitti, R. M. et al., refer to the new molecule as Apo-2 ligand or (Apo-2L). For convenience, it will be referred to herein as TRAIL. The amino acid sequence of TRAIL is given in SEQ ID NO: 72.

[0012] Unlike FAS ligand whose transcripts appear to be largely restricted to stimulated T-cells, significant levels of TRAIL are seen in many tissues, and it is constitutively transcribed by some cell lines. It has been shown that TRAIL acts independently from FAS ligand (Wiley, S. R., et al. (1995)), supra). Studies by Marsters, S. A. et al., have indicated that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signalling by FAS/Apo-1L but much faster than TNF-induced apoptosis (Current Biology, 6:750-752 (1996)).

[0013] As many as five TRAIL receptors have been identified, including TR7 (also known as TRAIL receptor 1 (TRAIL-R1) and death receptor 4 (DR4), Pan et al., Science 276:111-3 (1997), International Patent Application Nos. WO98/32856, WO/0067793, WO99/37684, WO2000/34355, WO99/02653, SEQ ID NO: 1); TR7 (also referred to as TRAIL receptor 2 (TRAIL-R2), DR5, and KILLER, Pan et al., Science 277:815-8 (1997), Sheridan et al., Science 277:818-21 (1997), Chaudhury et al., Immunity 7:821-30 (1997), International Patent Application Nos. WO98/46643, WO99/09165, WO99/11791, WO98/41629, WO/0066156, and WO98/35986, SEQ ID NO: 3); TR1 (also referred to as Osteoprotegrin (OPG) osteoclastogenesis inhibitory factor (OCIF), TNFRSF11B, and FTHMA-090 (International Patent Application Nos. WO98/12344, WO2000/54651, WO2001/04137, WO66/26217, WO98/07840, WO2000/21554, WO99/53942, and WO2001/03719, SEQ ID NO: 5); TR5 (also referred to as TRAIL receptor 3 (TRAIL-R3), decoy receptor 1 (DcR1) and TRID) (Degli-Esposti et al., J. Exp. Med. 186:1165-70 (1997), International Patent Application Nos. WO98/30693, WO/0071150, WO99/00423, EP867509, WO98/58062, SEQ ID NO: 2); and TR10 (also referred to as TRAIL Receptor 4 (TRAIL-R4), DcR2, and TRUNDD, Pan et al., FEBS Lett. 424:41-5 (1998), Degli-Eposti et al., Immunity 7:813-20 (1997), International Patent Application Nos. WO98/54202, WO/0073321, WO2000/08155, WO99/03992, WO 2000/34355 and WO9910484, SEQ ID NO: 4). TR7 and TR7 contain death domains in their cytoplasmic tails and the triggering of these receptors results in apoptosis. On the other hand, TR1, TR5 and TR10 can inhibit apoptosis induced by the cytotoxic ligand TRAIL in part because of their absent or truncated cytoplasmic death domains, respectively. Each of the publications and patents cited above is hereby incorporated by reference in their entireties, particularly with respect to the nucleotide and amino acid sequences of the TRAIL receptors disclosed therein.

[0014] The effects of TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of compositions, such as antibodies, that influence the biological activity of TNF receptors, both normally and in disease states. In particular, there is a need to isolate and characterize antibodies that modulate the biological activities of TRAIL receptors.

[0015] The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TR7 polypeptide or polypeptide fragment or variant of TR7. In particular, the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment or variant of human TR7 such as that of SEQ ID NO: 3. In some embodiments, an antibody of the invention that immunospecifically bind to a TR7 polypeptide, also bind TR7 (e.g., SEQ ID NO: 3), but not other proteins, including (TR1, TR5, and TR10 (SEQ ID NOs: 5, 2 and 4.)

[0016] The present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR7 or a fragment or variant thereof. In specific embodiments, the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with TR7 function or TR7 ligand function or aberrant TR7 or TR7 ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to a TR7 or a fragment or variant thereof. In highly preferred embodiments, the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma). Other diseases and disorders which can be treated, prevented or ameliorated with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes viral infections, and other viral infections) and proliferative disorders.

[0017] The present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR7 or a fragment or variant thereof. In specific embodiments, the present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with TR7 function or TR7 ligand function or aberrant TR7 or TR7 ligand expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TR7 or a fragment or variant thereof. In highly preferred embodiments, the present invention relates to antibody-based methods and compositions for detecting, diagnosing, or prognosing cancers and other hyperproliferative disorders (e.g., leukemia, carcinoma, and lymphoma). Other diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention include, but are not limited to, neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease), immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto's disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), infectious diseases (e.g., AIDS, herpes virus infections, and other viral infections), and proliferative disorders.

[0018] In highly preferred embodiments, antibodies of the present invention are used in methods and compositions for preventing, diagnosing, prognosing, treating or ameliorating the following types of cancer: breast cancer, lung cancer, (including non-small cell lung cancer), colon cancer, cancer of the urinary tract, bladder cancer, kidney cancer, pancreatic cancer, liver cancer, stomach cancer, prostate cancer, leukemia, Non-Hodgkin's lymphoma, esophageal cancer, brain cancer, leukemia, ovarian cancer, testicular cancer, melanoma, uterine cancer, cervical cancer, cancer of the larynx, rectal cancer, and cancers of the oral cavity. In specific embodiments, antibodies of the invention are administered in combination with chemotherapeutics such as paclitaxel (Taxol), irinotecan (Camptosar, CPT-11), irinotecan analogs, and gemcitabine (GEMZAR)).

[0019] Another embodiment of the present invention includes the use of the antibodies of the invention as a diagnostic tool to monitor the expression of TR7 expression on cells.

[0020] The present inventors have generated single chain Fv's (scFvs) that immunospecifically bind TR7 polypeptides (e.g., SEQ ID NO: 3). Thus, the invention encompases these scFvs, listed in Table 1. In addition, the invention encompasses cell lines engineered to express antibodies corresponding to these scFvs which are deposited with the American Type Culture Collection (ATCC) as of the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1 The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

[0021] Further, the present invention encompasses polynucleotides encoding the scFvs, as well as the amino acid sequences of the scFvs. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of the scFvs referred to in Table 1), that immunospecifically bind to TR7 or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules. In highly preferred embodiments, the present invention encompasses antibodies, or fragments or variants thereof, that bind to the extracellular regions/domains of TR7 or fragments and variants thereof.

[0022] The present invention also provides antibodies that bind TR7 polypeptides which are coupled to a detectable label, such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label. The present invention also provides antibodies that bind TR7 polypeptides which are coupled to a therapeutic or cytotoxic agent. The present invention also provides antibodies that bind TR7 polypeptides which are coupled to a radioactive material.

[0023] The present invention also provides antibodies that bind TR7 polypeptides that act as either TR7 agonists or TR7 antagonists. In specific embodiments, the antibodies of the invention stimulate apoptosis of TR7 expressing cells. In other specific embodiments, the antibodies of the invention inhibit TRAIL binding to TR7. In other specific embodiments, the antibodies of the invention upregulate TR7 expression.

[0024] The present invention also provides antibodies that inhibit apoptosis of TR7 expressing cells. In other specific embodiments, the antibodies of the invention downregulate TR7 expression.

[0025] In further embodiments, the antibodies of the invention have a dissociation constant (K_D) of 10⁷ M or less. In preferred embodiments, the antibodies of the invention have a dissociation constant (K_D) of 10⁹ M or less.

[0026] The present invention further provides antibodies that stimulate apoptosis of TR7 expressing cells better than an equal concentration of TRAIL polypeptide stimulates apoptosis of TR7 expressing cells.

[0027] The present invention further provides antibodies that stimulate apoptosis of TR7 expressing cells equally well in the presence or absence of antibody cross-linking reagents; and/or stimulate apoptosis with equal or greater potency as an equal concentration of TRAIL in the absence of a cross-linking antibody or other cross-linking agent.

[0028] In further embodiments, antibodies of the invention have an off rate (k_off) of 10^3/sec or less. In preferred embodiments, antibodies of the invention have an off rate (k_off) of 10⁴/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (k_off) of 10⁵/sec or less.

[0029] The present invention also provides for antibodies that preferentially bind TR7 and/or TR7 relative to their ability to bind other proteins (including TRI, TR5 and TRIO).

[0030] In certain embodiments, properties of the antibodies of the present invention, as detailed in the Examples below, make the antibodies better therapeutic agents than previously described TR7 binding antibodies.

[0031] The present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab)₂ fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

[0032] The present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

[0033] The present invention also provides for a nucleic acid molecule(s), generally isolated, encoding an antibody (including molecules, such as scFvs, VH domains, or VL domains, that comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention. The present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof. The present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention. The present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule. These and other aspects of the invention are described in further detail below.

[0034] FIGS. 1A-C show the the ability of anti-TR7 antibodies to induce apoptosis of MD-MBA-231 and SW480 cells in vitro in the presence and absence of cycloheximide.

[0035] FIG. 2 shows the effect of anti-TRAIL Receptor antibody treatment on SW480 tumor formation in Swiss nu/nu mice.

[0036] Definitions

[0037] The term antibody, as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term antibody herein include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab fragments, F(ab)₂, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term single chain Fv or scFv as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody. Antibodies that immunospecifically bind to TR7 may have cross-reactivity with other antigens, e.g., another TRAIL Receptor. Preferably, antibodies that immunospecifically bind to TR7 do not cross-react with other antigens (e.g., other TRAIL receptors or other members of the Tumor Necrosis Factor Receptor superfamily). Antibodies that immunospecifically bind to TR7 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0038] Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab) fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.

[0039] Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab)₂ fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate and SATA N-succinimidyl S-acethylthio-acetate (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

[0040] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94., both of which are hereby incorporated by reference in their entireties.) ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0041] Unless otherwise defined in the specification, specific binding or immunospecifc binding by an anti-TR7 antibody means that the anti-TR7 antibody binds TR7 but does not significantly bind to (i.e., cross react with) proteins other than TR7, such as other proteins in the same family of proteins). An antibody that binds TR7 protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the TR7-specific antibody of the invention preferentially binds TR7 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that TR7-specific antibodies bind to epitopes of TR7, an antibody that specifically binds TR7 may or may not bind fragments of TR7 and/or variants of TR7 (e.g., proteins that are at least 90% identical to TR7) depending on the presence or absence of the epitope bound by a given TR7-specific antibody in the TR7 fragment or variant. Likewise, TR7-specific antibodies of the invention may bind species orthologues of TR7 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, TR7-specific antibodies of the invention may bind modified forms of TR7, for example, TR7 fusion proteins. In such a case when antibodies of the invention bind TR7 fusion proteins, the antibody must make binding contact with the TR7 moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to TR7 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0042] In some embodiments the present invention encompasses antibodies that immunospecifcally or specifically bind both TR7 and TR4. Specific binding or immunospecifc binding by an antibody that immunospecifically binds TR7 and TR4 means that the antibody binds TR7 and TR4 but does not significantly bind to (i.e., cross react with) proteins other than TR7 or TR4, such as other proteins in the same family of proteins). An antibody that binds TR7 and TR4 proteins and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the antibody that immunospcifically or specifically binds both TR7 and TR4 preferentially binds TR7 and TR4 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that antibodies that bind TR7 and TR4 bind to epitopes common to TR7 and TR4, an antibody that specifically binds TR7 and TR4 may or may not bind fragments of TR7, TR4 and/or variants of TR7 or TR4 (e.g., proteins that are at least 90% identical to TR7 or TR4, respectively) depending on the presence or absence of the epitope bound by a given antibody in the TR7 or TR4 fragment or variant. Likewise, antibodies of the invention that immunospecifically bind TR7 and TR4 may bind species orthologues of TR7 and/or TR4 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologues. Additionally, antibodies of the invention that immunospecifically bind TR7 and TR4 may bind modified forms of TR7 or TR4, for example, TR7 or TR4 fusion proteins. In such a case when antibodies of the invention bind fusion proteins, the antibody must make binding contact with the TR7 or TR4 moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to TR7 or TR4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

[0043] The term variant as used herein refers to a polypeptide that possesses a similar or identical function as a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof, or possess a similar or identical structure of a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof, respectively. A variant having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of TR7 polypeptide (SEQ ID NO: 3) or a fragment thereof, an anti-TR7 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding TR7 (SEQ ID NO: 3), a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1), described herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scfvs referred to in Table 1), described herein. A polypeptide with similar structure to a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a TR7 polypeptide, a fragment of a TR7 polypeptide, an anti-TR7 antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

[0044] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identitynumber of identical overlapping positions/total number of positions100%). In one embodiment, the two sequences are the same length.

[0045] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268(1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877(1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410(1990) have incorporated such an alogrithm. BLAST nucleotide searches can be performed with the BLASTn program (score100, wordlength12) to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program (score50, wordlength3) to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402(1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. (See http://www.ncbi.nlm.nih.gov.)

[0046] Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an alogrithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10 :3-5(1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8(1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0047] The term derivative as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a TR7 polypeptide, a fragment of a TR7 polypeptide, or an antibody of the invention that immunospecifically binds to a TR7 polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term derivative as used herein also refers to a TR7 polypeptide, a fragment of a TR7 polypeptide, or an antibody that immunospecifically binds to a TR7 polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a TR7 polypeptide, a fragment of a TR7 polypeptide, or an anti-TR7 antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a TR7 polypeptide, a fragment of a TR7 polypeptide, or an anti-TR7 antibody, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a TR7 polypeptide, a fragment of a TR7 polypeptide, or an anti-TR7 antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a TR7 polypeptide, a fragment of a TR7 polypeptide, or an anti-TR7 antibody, described herein.

[0048] The term epitopes as used herein refers to portions of TR7 having antigenic or immunogenic activity in an animal, preferably a mammal. An epitope having immunogenic activity is a portion of TR7 that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of TR7 to which an antibody immunospecifically binds as determined by any method known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic.

[0049] The term fragment as used herein refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of TR7, or an anti-TR7 antibody (including molecules such as scfv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to TRAIL receptor.

[0050] The term fusion protein as used herein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-TR7 antibody of the invention and an amino acid sequence of a heterologous polypeptide (e.g., a polypeptide unrelated to an antibody or antibody domain).

[0051] The term host cell as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0052] Antibodies of the present invention are preferably provided in an isolated form, and preferably are substantially purified. By isolated is intended an antibody removed from its native environment. Thus, for eaxample, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

[0053] By isolated antibody is intended an antibody removed from its native environment. Thus, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

[0054] Antibody Structure

[0055] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, lgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

[0056] Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

[0057] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the heavy and the ligt chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

[0058] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547 1553 (1992). In addition, bispecific antibodies may be formed as diabodies (Holliger et al. Diabodies: small bivalent and bispecific antibody fragments PNAS USA 90:6444-6448 (1993)) or Janusins (Traunecker et al. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells EMBO J 10:3655-3659 (1991) and Traunecker et al. Janusin: new molecular design for bispecific reagents Int J Cancer Suppl 7:51-52 (1992)).

[0059] Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab, and Fv).

[0060] Anti-TR7 Antibodies

[0061] Using phage display technology, single chain antibody molecules (scFvs) that immunospecifically bind to TR7 (or fragments or variants thereof) have been identified. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind to TR7 (or fragments or variants thereof) are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

[0062] In particular, the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-56, preferably SEQ ID NOS: 42, 50, and 56 as referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind to TR7 are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules (e.g., SEQ ID NOs: 57-71).

[0063] ScFvs corresponding to SEQ ID NOs: 42-56 were selected for their ability bind TR7 polypeptide.

[0064] The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of TR7. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1. Such scFvs may routinely be converted to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 4 below.

[0065] NS0 cell lines that express IgG1 antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (ATCC) on the dates listed in Table 1 and given the ATCC Deposit Numbers identified in Table 1. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure.

[0066] Accordingly, in one embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.

[0067] In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO TR7 2521 140 p:12 deposited with the ATCC on Mar. 25, 2002 and given ATCC Deposit Number PTA-4178 (See Table 1).

[0068] In another preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO TR7 2521 (5G08) 176-41, p:10 deposited with the ATCC on Jul. 10, 2002 and given ATCC Deposit Number PTA-4539 (See Table 1).

[0069] In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO TR7 2654 (84A02) 62 p:10 deposited with the ATCC on May 21, 2002 and given ATCC Deposit Number PTA-4376 (See Table 1).

[0070] In another preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO TR7 Ab 2834 10, p12 deposited with the ATCC on Jul. 17, 2002 and given ATCC Deposit Number PTA-4547 (See Table 1).

TABLE 1
scFvs that Immunospecifically bind to TRAIL Receptors
	scFv
	pro-	scfv
	tein	DNA									Cell Line
	SEQ	SEQ	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	AAs of	Ex-	ATCC	ATCC
	ID	ID	VH	VH	VH	VH	VL	VL	VL	VL	pressing	Deposit	Deposit
ScFv	NO:	NO:	Domain	CDR1	CDR2	CDR3	Domain	CDR1	CDR2	CDR3	antibody	Number	Date
CM005G08	42	57	1-121	26-35	50-66	99-110	136-244	158-168	184-190	223-233	NSO TR7	PTA-4178	Mar. 25,
											2521 140		2002
											p; 12
											NSO TR7	PTA-4539	Jul. 10,
											2521		2002
											(5GOB)
											176-41,
											p: 10
CM005A08	43	58	1-122	26-35	50-66	99-111	137-245	159-169	185-191	224-234
CM014C10	44	59	1-119	26-35	50-66	99-108	136-246	158-170	186-192	225-235
CM029B01	45	60	1-121	26-35	50-66	99-110	136-244	158-168	184-190	223-233
CM033D06	46	61	1-114	26-35	50-66	99-103	129-235	151-161	177-183	216-224
CM013A11	47	62	1-121	26-35	50-66	99-110	139-245	161-171	187-193	226-234
CM013F04	48	63	1-120	26-35	50-66	99-109	137-247	159-172	188-194	227-236
CM088F10	49	64	1-128	26-35	50-63	99-117	145-255	167-180	196-202	235-244
CM084A02	50	65	1-120	26-35	50-66	99-109	137-248	159-171	187-193	226-237	NSO TR7	PTA-4376	May 21,
											2654		2002
											(84A02)
											62 p: 10
CM087C06	51	66	1-116	26-35	50-66	99-105	133-243	155-167	183-189	222-232
CM055A01	52	67	1-117	26-35	50-66	99-106	133-240	154-164	180-186	219-229
CM085C11	53	68	1-116	26-35	50-66	99-105	133-243	155-167	183-189	222-232
CM089A03	54	69	1-126	26-35	50-66	99-115	143-253	165-178	194-200	233-242
CM075A01	55	70	1-115	26-35	50-66	99-104	133-243	155-167	183-189	224-231
CM059H03	56	71	1-121	26-35	50-66	99-110	139-247	162-173	189-195	228-236	NSO TR7	PTA-4547	Jul. 17,
											Ab 2834		2002
											10 p12

[0071] The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a TR7 polypeptide or a fragment, variant, or fusion protein thereof. A TR7 polypeptide includes, but is not limited to, TR7 (SEQ ID NO: 3) or the polypeptide encoded by the cDNA in clone HLYBX88 contained in ATCC Deposit 97920 deposited Mar. 7, 1997. In some embodiments, antibodies of the present invention may immunospecificallybind to both TR7 as described above and to TR4 (SEQ ID NO: 1) or the polypeptide encoded by the cDNA in clone HCUDS60 contained in ATCC Deposit 97853 deposited Jan. 21, 1997. TRAIL receptors may be produced through recombinant expression of nucleic acids encoding the polypeptides of SEQ ID NOs: 1-5, (TR4, TR5, TR7, TR10, and TR1, respectively (e.g., the cDNAs in the ATCC Deposit Numbers 97853, (TR4) 97798 (TR5, deposited Nov. 20, 1996), 97920 (TR7), or 209040 (TR10, deposited May 15, 1997).

[0072] In one embodiment, the antibodies of the invention preferentially bind TR7 (SEQ ID NO: 3), or fragments, variants, or fusion proteins thereof (e.g., the extracellular region of TR7 fused to an Fc domain) relative to their ability to bind other proteins including TR1, TR4, TR5 or TR10 (SEQ ID NOs: 5, 1, 2 and 4) or fragments, variants, or fusion proteins thereof. In other preferred embodiments, the antibodies of the invention preferentially bind to TR7 and TR4 (SEQ ID NOs: 3 and 1), or fragments and variants thereof relative to their ability to bind other proteins including TR1, TR5 or TR10 (SEQ ID NOs: 5, 2 and 4) or fragments, variants, or fusion proteins thereof. In other preferred embodiments, the antibodies of the invention bind TR1 TR4, TR5, TR7 and TR10 (SEQ ID NOs: 5, 1, 2, 3, and 4). An antibody's ability to preferentially bind one antigen compared to another antigen may be determined using any method known in the art.

[0073] TR7 Polypetides

[0074] In certain embodiments of the present invention, the antibodies of the present invention bind TR7 polypeptide, or fragments or variants thereof. The following section describes the TR7 polypeptides, fragments and variants that may be bound by the antibodies of the invention in more detail. The TR7 polypeptides, fragments and variants which may be bound by the antibodies of the invention are also described in, for example, International Publication Numbers WO98/41629, WO/0066156, and WO98/35986 which are herein incorporated by reference in their entireties.

[0075] In certain embodiments, the antibodies of the present invention immunospecifically bind TR7 polypeptide. An antibody that immunospecifically binds TR7 may, in some embodiments, bind fragments, variants (including species orthologs of TR7), multimers or modified forms of TR7. For example, an antibody immunospecific for TR7 may bind the TR7 moiety of a fusion protein comprising all or a portion of TR7.

[0076] TR7 proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to antibodies that bind TR7 proteins found as monomers or as part of multimers. In specific embodiments, the TR7 polypeptides are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

[0077] Antibodies of the invention may bind TR7 homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only TR7 proteins of the invention (including TR7 fragments, variants, and fusion proteins, as described herein). These homomers may contain TR7 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR7 proteins having an identical polypeptide sequence. In another specific embodiment, antibodies of the invention bind TR7 homomers containing TR7 proteins having different polypeptide sequences. In specific embodiments, antibodies of the invention bind a TR7 homodimer (e.g., containing TR7 proteins having identical or different polypeptide sequences). In additional embodiments, antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer of TR7.

[0078] In specific embodiments antibodies of the presnt invention bind TR7 homotrimers (e.g., containing TR7 proteins having identical or different polypeptide sequences).

[0079] As used herein, the term heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to TR7 polypeptides sequences) in addition to the TR7 proteins of the invention. In a specific embodiment, antibodies of the invention bind a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer containing one or more TR7 polypeptides.

[0080] In specific embodiments antibodies of the presnt invention bind a TR7 heterotrimer (e.g., containing 1 or 2 TR7 proteins and 2 or 1, respectively, TR4 proteins).

[0081] Multimers bound by one or more antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers bound by one or more antibodies of the invention, such as, for example, homodimers or homotrimers, are formed when TR7 proteins contact one another in solution. In another embodiment, heteromultimers bound by one or more antibodies of the invention, such as, for example, heterotrimers or heterotetramers, are formed when TR7 proteins contact antibodies to TR7 polypeptides (or antibodies to the heterologous polypeptide sequence in a fusion protein) in solution. In other embodiments, multimers bound by one or more antibodies of the invention are formed by covalent associations with and/or between the TR7 proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO: 3 or the polypeptide encoded by the deposited cDNA clone of ATCC Deposit 97920). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR7 fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a TR7-Fc fusion protein (as described herein). In another specific example, covalent associations of fusion proteins are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).

[0082] The multimers that may be bound by one or more antibodies of the invention may be generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers that may be bound by one or more antibodies of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins that may be bound by one or more antibodies of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer that may be bound by one or more antibodies of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

[0083] Alternatively, multimers that may be bound by one or more antibodies of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers that may be bound by one or more antibodies of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer that may be bound by one or more antibodies of the invention are generated by ligating a polynucleotide sequence encoding a TR7 polypeptide to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant TR7 polypeptides which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, two or more TR7 polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR7 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology. In specific embodiments, antibodies of the invention bind proteins comprising multiple TR7 polypeptides separated by peptide linkers.

[0084] Another method for preparing multimer TR7 polypeptides involves use of TR7 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR7 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble TR7 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR7 is recovered from the culture supernatant using techniques known in the art. In specific embodiments, antibodies of the invention bind TR7-leucine zipper fusion protein monomers and/or TR7-leucine zipper fusion protein multimers.

[0085] Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993). Thus, trimeric TR7 may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. In specific embodiments, antibodies of the invention bind TR7-leucine zipper fusion protein trimers.

[0086] Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TR7. In specific embodiments, antibodies of the invention bind TR7-fusion protein monomers and/or TR7 fusion protein trimers.

[0087] Antibodies of the invention that bind TR7 receptor polypeptides may bind them as isolated polypeptides or in their naturally occurring state. By isolated polypeptide is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also, intended as an isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the TR7 polypeptide is substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Thus, antibodies of the present invention may bind recombinantly produced TR7 receptor polypeptides. In a specific embodiment, antibodies of the present invention bind a TR7 receptor expressed on the surface of a cell, wherein said TR7 polypeptide is encoded by a polynucleotide encoding amino acids 1 to 411 of SEQ ID NO: 3 operably associated with a regulatory sequence that controlsexpression of said polynucleotide.

[0088] Antibodies of the present invention may bind TR7 polypeptides or polypeptide fragments including polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO: 3, encoded by the cDNA contained in ATCC deposit Number 97920, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the ATCC deposit Number 97920, or the complementary strand thereto. Protein fragments may be free-standing, or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Antibodies of the present invention may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 51, 52 to 78, 79 to 91, 92 to 111, 112 to 134, 135 to 151, 152 to 178, 179 to 180, 181 to 208, 209 to 218, 219 to 231, 232 to 251,252 to 271, 272 to 291, 292 to 311, 312 to 323, 324 to 361, 362 to 391, 392 to 411 of SEQ ID NO: 3. In this context about includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Moreover, polypeptide fragments can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context about includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.

[0089] Preferred polypeptide fragments of the present invention include a member selected from the group: a polypeptide comprising or alternatively, consisting of, the TR7 receptor extracellular domain (predicted to constitute amino acid residues from about 52 to about 184 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, both TR7 cysteine rich domains (both of which may be found in the protein fragment consisting of amino acid residues from about 84 to about 179 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, the TR7 cysteine rich domain consisting of amino acid residues from about 84 to about 131 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, the TR7 cysteine rich domain consisting of amino acid residues from about 132 to about 179 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, the TR7 receptor transmembrane domain (predicted to constitute amino acid residues from about 185 to about 208 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, fragment of the predicted mature TR7 polypeptide, wherein the fragment has a TR7 functional activity (e.g., antigenic activity or biological acitivity); a polypeptide comprising or alternatively, consisting of, the TR7 receptor intracellular domain (predicted to constitute amino acid residues from about 209 to about 411 in SEQ ID NO: 3); a polypeptide comprising or alternatively, consisting of, the TR7 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; a polypeptide comprising, or alternatively consisting of, the TR7 receptor death domain (predicted to constitute amino acid residues from about 324 to about 391 in SEQ ID NO: 3); and a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the TR7 receptor protein. In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively, consist of, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members. As above, with the leader sequence, the amino acid residues constituting the TR7 receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, as one of ordinary skill would appreciate, the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.

[0090] As discussed above, it is believed that one or both of the extracellular cysteine rich motifs of TR7 is important for interactions between TR7 and its ligands (e.g., TRAIL). Accordingly, in highly preferred embodiments, antibodies of the present invention bind TR7 polypeptide fragments comprising, or alternatively consisting of, amino acid residues 84 to 131, and/or 132 to 179 of SEQ ID NO: 3. In another highly preferred embodiment, antibodies of the present invention bind TR7 polypeptides comprising, or alternatively consisting of, both of the extracellular cysteine rich motifs (amino acid residues 84 to 179 of SEQ ID NO: 3.) In another preferred embodiment, antibodies of the present invention bind TR7 polypeptides comprising, or alternatively consisting the extracellular soluble domain of TR7 (amino acid residues 52 to 184 of SEQ ID NO: 2.) In other highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR7 (e.g., one or both cysteine rich domains) agonize the TR7 receptor.

[0091] In other highly preferred embodiments, the antibodies of the invention that bind all or a portion of the extracellular soluble domain of TR7 (e.g., one or both cysteine rich domains) induce cell death of the cell expressing the TR7 receptor.

[0092] Antibodies of the invention may also bind fragments comprising, or alternatively, consisting of structural or functional attributes of TR7. Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions (alpha-regions), beta-sheet and beta-sheet-forming regions (beta-regions), turn and turn-forming regions (turn-regions), coil and coil-forming regions (coil-regions), hydrophillic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., regions of polypeptides consisting of amino acid residues having an antigenic index of or equal to greater than 1.5, as identified using the default parameters of the Jameson-Wolf program) of TR7. Certain preferred regions are those disclosed in Table 2 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence of SEQ ID NO: 3, such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic regions and Hopp-Woods predicted hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.

[0093] The data representing the structural or functional attributes of TR7 set forth in Table 2, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. Column I represents the results of a Garnier-Robson analysis of alpha helical regions; Column II represents the results of a Chou-Fasman analysis of alpha helical regions; Column III represents the results of a Garnier Robson analysis of beta sheet regions; Column IV represents the results of a Chou-Fasman analysis of beta sheet regions; Column V represents the results of a Garnier Robson analysis of turn regions; Column VI represents the results of a Chou-Fasman analysis of turn regions; Column VII represents the results of a Garnier Robson analysis of coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column IX represents a Hopp-Woods hydrophobicity plot; Column X represents the results of an Eisenberg analysis of alpha amphipathic regions; Column XI represents the results of an Eisenberg analysis of beta amphipathic regions; Column XII represents the results of a Karplus-Schultz analysis of flexible regions; Column XIII represents the Jameson-Wolf antigenic index score; and Column XIV represents the Emini surface probability plot.

[0094] In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of TR7 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response. The columns in Table 2 present the result of different analyses of the TR7 protein sequence.

[0095] The above-mentioned preferred regions set out in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO: 3. As set out in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Preferably, antibodies of the present invention bind TR7 polypeptides or TR7 polypeptide fragments and variants comprising regions of TR7 that combine several structural features, such as several (e.g., 1, 2, 3, or 4) of the same or different region features set out above and in Table 2.

TABLE 2
Res	Position	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII	XIV
Met	1	A	.	.	.	.	.	.	1.11	0.70	.	*	.	1.29	2.18
Glu	2	A	.	.	.	.	.	.	1.50	0.70	.	*	.	1.63	1.69
Gln	3	A	.	.	.	.	T	.	1.89	0.73	.	*	.	2.17	2.28
Arg	4	.	.	.	.	T	T	.	1.69	0.76	.	*	.	2.91	3.71
Gly	5	.	.	.	.	T	T	.	1.87	0.87	.	*	F	3.40	2.17
Gln	6	.	.	.	.	T	T	.	1.88	0.44	.	*	F	2.76	1.93
Asn	7	.	.	.	.	.	.	C	1.29	0.34	.	*	F	1.87	1.00
Ala	8	.	.	.	.	.	.	C	0.99	0.16	.	.	F	1.08	1.02
Pro	9	.	.	.	.	.	.	C	0.53	0.11	.	*	.	0.44	0.79
Ala	10	A	.	.	.	.	.	.	0.29	0.14	.	*	.	0.10	0.48
Ala	11	A	.	.	.	.	T	.	0.40	0.24	.	.	.	0.10	0.48
Ser	12	A	.	.	.	.	T	.	0.44	0.26	.	*	F	0.85	0.61
Gly	13	A	.	.	.	.	T	.	1.14	0.69	.	*	F	1.30	1.22
Ala	14	A	.	.	.	.	T	.	1.32	1.19	.	*	F	1.30	2.36
Arg	15	A	.	.	.	T	.	.	1.57	1.19	.	*	F	1.50	2.39
Lys	16	.	.	.	.	T	.	.	1.94	1.14	.	.	F	1.50	2.39
Arg	17	.	.	.	.	T	.	.	1.90	1.14	.	*	F	1.80	3.66
His	18	.	.	.	.	.	.	C	2.03	1.21	*	*	F	1.90	1.85
Gly	19	.	.	.	.	.	T	C	2.73	0.79	*	*	F	2.40	1.43
Pro	20	.	.	.	.	.	T	C	2.62	0.79	*	*	F	2.70	1.43
Gly	21	.	.	.	.	.	T	C	1.99	0.79	*	.	F	3.00	1.82
Pro	22	.	.	.	.	.	T	C	1.99	0.79	.	*	F	2.70	1.86
Arg	23	.	A	.	.	.	.	C	1.68	1.21	*	.	F	2.30	2.35
Glu	24	.	A	B	.	.	.	.	1.43	1.21	*	.	F	2.10	2.35
Ala	25	.	A	.	.	T	.	.	1.76	1.14	*	.	F	2.50	1.54
Arg	26	.	A	.	.	T	.	.	1.89	1.57	*	.	F	2.50	1.54
Gly	27	.	.	.	.	T	.	.	1.76	1.14	*	.	F	3.00	1.37
Ala	28	.	.	.	.	T	.	C	1.43	0.71	*	*	F	2.70	1.35
Arg	29	.	.	.	.	.	T	C	1.54	0.79	*	*	F	2.66	1.06
Pro	30	.	.	.	.	.	T	C	1.28	0.79	*	*	F	2.62	2.10
Gly	31	.	.	.	.	.	T	C	0.96	0.57	*	*	F	2.58	1.54
Pro	32	.	.	.	.	.	T	C	1.34	0.64	*	*	F	2.54	1.22
Arg	33	.	.	.	.	.	.	C	1.62	0.64	*	*	F	2.60	1.58
Val	34	.	.	.	.	.	.	C	0.70	0.59	*	*	F	2.34	2.30
Pro	35	.	.	B	.	.	.	.	0.06	0.33	*	*	F	1.58	1.23
Lys	36	.	.	B	B	.	.	.	0.41	0.11	*	.	F	0.97	0.46
Thr	37	.	.	B	B	.	.	.	1.06	0.57	*	*	F	0.19	0.52
Leu	38	.	.	B	B	.	.	.	2.02	0.57	*	*	.	0.60	0.25
Val	39	.	.	B	B	.	.	.	1.76	0.79	.	.	.	0.60	0.09
Leu	40	A	.	.	B	.	.	.	2.13	1.29	.	.	.	0.60	0.06
Val	41	A	.	.	B	.	.	.	3.03	1.30	.	.	.	0.60	0.08
Val	42	A	.	.	B	.	.	.	3.53	1.26	.	.	.	0.60	0.08
Ala	43	A	.	.	B	.	.	.	3.53	1.30	.	.	.	0.60	0.08
Ala	44	A	.	.	B	.	.	.	3.49	1.30	.	.	.	0.60	0.09
Val	45	A	.	.	B	.	.	.	3.53	1.34	.	.	.	0.60	0.10
Leu	46	A	.	.	B	.	.	.	2.98	1.34	.	.	.	0.60	0.07
Leu	47	A	.	.	B	.	.	.	2.71	1.23	.	.	.	0.60	0.09
Leu	48	A	.	.	B	.	.	.	2.12	1.23	.	.	.	0.60	0.13
Val	49	A	.	.	B	.	.	.	1.83	0.59	.	.	.	0.60	0.27
Ser	50	A	.	.	B	.	.	.	1.57	0.29	.	*	.	0.30	0.44
Ala	51	A	A	.	.	.	.	.	1.57	0.10	.	.	.	0.30	0.54
Glu	52	A	A	.	.	.	.	.	1.64	0.10	.	.	.	0.30	0.60
Ser	53	A	A	.	B	.	.	.	1.14	0.14	.	.	.	0.30	0.31
Ala	54	A	A	.	B	.	.	.	0.29	0.24	.	.	.	0.30	0.45
Leu	55	A	A	.	B	.	.	.	0.01	0.14	.	.	.	0.30	0.45
Ile	56	A	A	.	B	.	.	.	0.60	0.54	.	.	.	0.60	0.58
Thr	57	A	A	.	B	.	.	.	0.21	0.16	.	.	F	0.15	0.96
Gln	58	A	A	.	B	.	.	.	0.50	0.34	.	.	F	0.15	0.96
Gln	59	A	A	.	B	.	.	.	0.12	0.16	.	.	F	0.00	1.38
Asp	60	.	A	.	B	T	.	.	0.69	0.10	.	.	F	1.00	1.48
Leu	61	.	A	.	.	.	.	C	1.58	0.19	.	*	F	0.80	1.48
Ala	62	.	A	.	.	.	.	C	2.00	0.19	.	*	F	0.80	1.48
Pro	63	.	A	.	.	.	.	C	1.41	0.59	.	*	F	1.10	1.73
Gln	64	.	A	.	.	T	.	.	0.82	0.09	.	*	F	1.00	2.13
Gln	65	A	A	.	.	.	.	.	0.61	0.27	.	*	F	0.60	2.13
Arg	66	A	A	.	.	.	.	.	1.42	0.34	.	*	F	0.60	2.13
Ala	67	A	A	.	.	.	.	.	2.01	0.37	.	*	F	0.94	2.13
Ala	68	A	A	.	.	.	.	.	2.27	0.37	*	*	F	1.28	2.13
Pro	69	A	A	.	.	.	.	.	2.38	0.77	*	*	F	1.92	2.17
Gln	70	.	A	.	.	T	.	.	2.08	0.77	*	.	F	2.66	4.21
Gln	71	.	.	.	.	T	T	.	1.67	0.89	*	*	F	3.40	5.58
Lys	72	.	.	.	.	T	T	.	2.04	1.00	.	.	F	3.06	4.84
Arg	73	.	.	.	.	T	T	.	2.33	1.00	.	.	F	2.97	4.32
Ser	74	.	.	.	.	.	T	C	2.54	1.01	.	.	F	2.68	3.34
Ser	75	.	.	.	.	.	T	C	2.20	1.41	.	.	F	2.59	2.89
Pro	76	.	.	.	.	T	T	.	1.39	0.99	.	.	F	2.70	1.46
Ser	77	.	.	.	.	T	T	.	0.68	0.30	.	.	F	2.50	0.90
Glu	78	.	.	.	.	T	T	.	0.36	0.11	.	*	F	2.25	0.36
Gly	79	.	.	.	.	T	.	.	0.44	0.07	.	.	F	1.80	0.36
Leu	80	.	.	.	.	T	.	.	0.40	0.07	.	.	F	1.55	0.42
Cys	81	.	.	.	.	.	.	C	0.58	0.03	.	.	.	0.95	0.24
Pro	82	.	.	.	.	.	T	C	0.84	0.47	*	.	F	0.15	0.33
Pro	83	.	.	.	.	T	T	.	0.04	0.54	*	.	F	0.35	0.54
Gly	84	.	.	.	.	T	T	.	0.00	0.54	*	.	.	0.20	0.70
His	85	.	.	.	.	.	T	C	0.81	0.36	*	.	.	0.30	0.61
His	86	.	.	.	.	.	.	C	1.48	0.07	*	.	.	0.70	0.68
Ile	87	.	.	.	.	.	.	C	1.34	0.50	*	*	.	1.19	1.15
Ser	88	.	.	.	.	.	.	C	1.67	0.50	*	*	F	1.53	0.84
Glu	89	.	.	.	.	T	.	.	2.01	1.00	*	*	F	2.52	1.21
Asp	90	.	.	.	.	T	.	.	1.38	1.50	*	*	F	2.86	2.88
Gly	91	.	.	.	.	T	T	.	0.52	1.61	*	*	F	3.40	1.15
Arg	92	.	.	.	.	T	T	.	1.11	1.31	*	*	F	2.91	0.47
Asp	93	.	.	.	.	T	T	.	0.74	0.93	.	*	F	2.57	0.37
Cys	94	.	.	.	.	T	T	.	0.79	0.36	.	*	.	1.78	0.20
Ile	95	.	.	.	.	T	.	.	0.54	0.79	.	*	.	1.54	0.21
Ser	96	.	.	.	.	T	.	.	0.54	0.03	.	*	.	1.18	0.19
Cys	97	.	.	.	.	T	T	.	0.43	0.40	.	*	.	0.76	0.36
Lys	98	.	.	.	.	T	T	.	0.43	0.23	.	.	.	1.34	0.88
Tyr	99	.	.	.	.	T	T	.	0.86	0.46	.	*	F	2.52	1.10
Gly	100	.	.	.	.	T	T	.	1.44	0.09	.	*	F	2.80	3.22
Gln	101	.	.	.	.	T	T	.	1.43	0.27	*	.	F	2.52	2.16
Asp	102	.	.	.	.	T	T	.	2.07	0.21	*	*	F	1.64	1.99
Tyr	103	.	.	.	.	T	T	.	1.73	0.04	*	*	F	1.96	2.73
Ser	104	.	.	.	.	T	T	.	1.98	0.44	*	.	F	0.78	1.66
Thr	105	.	.	.	.	T	.	.	2.32	0.44	*	.	F	0.30	1.60
His	106	.	.	.	.	T	.	.	1.51	0.44	*	.	.	0.15	1.70
Trp	107	.	.	.	.	T	T	.	0.70	0.37	*	.	.	0.65	1.05
Asn	108	.	.	.	.	T	T	.	0.24	0.67	.	.	.	0.20	0.60
Asp	109	.	.	.	.	T	T	.	0.12	0.97	*	.	.	0.20	0.38
Leu	110	A	.	.	.	.	T	.	0.62	1.04	*	*	.	0.20	0.19
Leu	111	.	.	.	B	T	.	.	0.48	0.81	*	*	.	0.20	0.10
Phe	112	.	.	.	B	T	.	.	0.86	0.41	*	*	.	0.20	0.12
Cys	113	.	.	.	B	T	.	.	1.17	0.99	*	*	.	0.20	0.08
Leu	114	.	.	.	B	T	.	.	1.06	0.79	.	*	.	0.20	0.13
Arg	115	.	.	.	B	T	.	.	0.91	0.10	.	*	.	0.10	0.30
Cys	116	.	.	.	B	T	.	.	0.10	0.11	.	.	.	0.70	0.30
Thr	117	.	.	.	B	T	.	.	0.30	0.69	.	*	.	1.00	0.61
Arg	118	.	.	.	B	T	.	.	0.62	0.99	.	.	F	1.49	0.42
Cys	119	.	.	.	.	T	T	.	1.43	0.56	*	.	F	2.23	0.77
Asp	120	.	.	.	.	T	T	.	0.47	1.13	*	.	F	2.57	0.92
Ser	121	.	.	.	.	T	T	.	1.13	0.97	.	*	F	2.91	0.35
Gly	122	.	.	.	.	T	T	.	0.63	0.97	.	*	F	3.40	1.13
Glu	123	.	A	.	.	T	.	.	0.22	0.86	.	*	F	2.51	0.56
Val	124	A	A	.	.	.	.	.	0.68	0.47	.	*	F	1.47	0.56
Glu	125	.	A	.	.	T	.	.	0.01	0.43	.	*	.	1.38	0.87
Leu	126	.	A	.	.	T	.	.	0.00	0.29	.	*	.	1.04	0.27
Ser	127	.	.	.	.	.	T	C	0.03	0.20	.	*	F	0.45	0.52
Pro	128	.	.	.	.	T	T	.	0.28	0.04	.	*	F	0.93	0.44
Cys	129	.	.	.	.	T	T	.	0.69	0.53	.	*	F	0.91	0.77
Thr	130	.	.	.	.	T	T	.	0.69	0.16	.	*	F	2.24	1.12
Thr	131	.	.	.	.	T	.	.	1.19	0.14	.	*	F	2.32	1.16
Thr	132	.	.	.	.	T	T	.	0.63	0.09	.	*	F	2.80	3.13
Arg	133	.	.	.	.	T	T	.	0.18	0.01	.	.	F	2.52	1.61
Asn	134	.	.	.	.	T	T	.	0.84	0.07	.	.	F	1.49	0.60
Thr	135	.	.	.	.	T	T	.	0.49	0.01	.	.	F	1.81	0.72
Val	136	.	.	.	.	T	.	C	0.80	0.07	*	.	.	0.58	0.20
Cys	137	.	A	.	.	T	.	.	1.11	0.07	*	.	.	0.10	0.21
Gln	138	.	A	B	.	.	.	.	0.66	0.33	*	.	.	0.30	0.25
Cys	139	.	A	.	.	T	.	.	0.34	0.39	.	.	.	0.70	0.34
Glu	140	A	A	.	.	.	.	.	0.04	0.54	*	*	F	0.75	0.91
Glu	141	A	A	.	.	.	.	.	0.92	0.33	*	*	F	0.45	0.46
Gly	142	.	A	.	.	T	.	.	1.59	0.73	.	*	F	1.30	1.67
Thr	143	A	A	.	.	.	.	.	1.59	1.30	.	*	F	0.90	1.67
Phe	144	A	A	.	.	.	.	.	2.26	1.30	.	*	F	0.90	1.67
Arg	145	A	A	.	.	.	.	.	1.96	1.30	.	*	F	0.90	2.81
Glu	146	A	A	.	.	.	.	.	1.74	1.34	.	*	F	0.90	2.61
Glu	147	A	A	.	.	.	.	.	2.09	1.40	.	*	F	0.90	4.66
Asp	148	A	A	.	.	.	.	.	1.80	2.19	.	*	F	0.90	4.12
Ser	149	A	.	.	.	.	T	.	1.83	1.57	.	*	F	1.30	2.35
Pro	150	A	.	.	.	.	T	.	1.83	1.00	.	.	F	1.15	0.73
Glu	151	A	.	.	.	.	T	.	1.88	1.00	*	.	F	1.15	0.85
Met	152	A	.	.	.	.	T	.	1.21	1.00	*	*	.	1.49	1.28
Cys	153	A	.	.	.	.	T	.	1.32	0.81	*	*	.	1.68	0.44
Arg	154	A	.	.	.	.	T	.	1.31	1.24	*	.	.	2.02	0.50
Lys	155	.	.	.	.	T	T	.	1.18	0.76	*	*	F	2.91	0.73
Cys	156	.	.	.	.	T	T	.	0.51	0.94	*	.	F	3.40	1.35
Arg	157	.	.	.	.	T	.	.	0.90	0.94	*	.	F	2.71	0.37
Thr	158	.	.	.	.	T	.	.	1.68	0.51	*	.	F	2.37	0.28
Gly	159	.	.	.	.	T	.	.	1.22	0.51	*	.	F	2.43	1.04
Cys	160	.	.	.	.	.	T	C	0.58	0.66	.	*	F	2.19	0.53
Pro	161	.	.	.	.	T	T	.	0.39	0.04	.	*	F	2.00	0.36
Arg	162	.	.	.	.	T	T	.	0.32	0.11	.	*	F	1.65	0.27
Gly	163	.	.	.	.	T	T	.	0.22	0.31	*	*	.	2.50	1.01
Met	164	.	.	B	B	.	.	.	0.22	0.24	*	*	.	1.30	0.48
Val	165	.	.	B	B	.	.	.	0.44	0.24	*	*	.	1.30	0.24
Lys	166	.	.	B	B	.	.	.	0.01	0.24	*	*	.	1.30	0.41
Val	167	.	.	B	.	.	T	.	0.43	0.10	*	*	F	1.85	0.22
Gly	168	.	.	.	.	T	T	.	0.30	0.23	.	.	F	2.25	0.44
Asp	169	.	.	.	.	T	T	.	0.01	0.44	.	.	F	2.50	0.34
Cys	170	.	.	.	.	T	T	.	0.57	0.47	.	*	F	1.35	0.48
Thr	171	.	.	.	.	.	T	C	0.52	0.21	.	*	F	1.20	0.65
Pro	172	.	.	.	.	T	T	.	0.49	0.21	.	*	F	1.75	0.65
Trp	173	.	.	.	.	T	T	.	0.83	0.47	.	*	F	0.60	0.84
Ser	174	A	.	.	.	.	T	.	0.17	0.10	.	*	F	1.00	1.01
Asp	175	A	A	.	.	.	.	.	0.02	0.01	.	.	F	0.45	0.35
Ile	176	A	A	.	.	.	.	.	0.26	0.20	*	*	.	0.30	0.25
Glu	177	A	A	.	.	.	.	.	0.51	0.21	*	.	.	0.30	0.25
Cys	178	A	A	.	.	.	.	.	0.80	0.60	*	.	.	0.60	0.30
Val	179	A	A	.	.	.	.	.	0.80	0.60	*	*	.	0.60	0.74
His	180	A	A	.	.	.	.	.	0.46	0.90	.	*	.	0.60	0.58
Lys	181	A	A	.	.	.	.	.	0.46	0.47	*	.	F	0.60	1.06
Glu	182	A	.	.	.	.	T	.	0.43	0.36	*	.	F	1.00	1.00
Ser	183	A	.	.	.	.	T	.	0.66	0.31	.	.	F	0.85	0.52
Gly	184	A	.	.	.	T	T	.	0.14	0.13	.	.	F	1.25	0.18
Ile	185	A	.	.	.	.	T	.	0.97	0.30	.	.	.	0.10	0.10
Ile	186	.	.	B	B	.	.	.	1.32	0.94	.	*	.	0.60	0.06
Ile	187	.	.	B	B	.	.	.	2.18	1.04	.	.	.	0.60	0.08
Gly	188	.	.	B	B	.	.	.	2.47	1.26	.	*	.	0.60	0.09
Val	189	.	.	B	B	.	.	.	2.71	1.07	.	.	.	0.60	0.13
Thr	190	A	.	.	B	.	.	.	2.68	0.89	.	*	.	0.60	0.18
Val	191	A	.	.	B	.	.	.	2.64	0.84	.	.	.	0.60	0.14
Ala	192	A	.	.	B	.	.	.	2.57	1.06	.	*	.	0.60	0.14
Ala	193	A	.	.	B	.	.	.	3.11	1.10	.	.	.	0.60	0.08
Val	194	A	.	.	B	.	.	.	3.11	1.30	.	.	.	0.60	0.07
Val	195	A	.	.	B	.	.	.	3.39	1.30	.	.	.	0.60	0.05
Leu	196	A	.	.	B	.	.	.	3.39	1.30	.	.	.	0.60	0.05
Ile	197	A	.	.	B	.	.	.	3.50	1.44	.	.	.	0.60	0.05
Val	198	A	.	.	B	.	.	.	3.77	1.59	.	.	.	0.60	0.06
Ala	199	A	.	.	B	.	.	.	3.58	1.59	.	.	.	0.60	0.06
Val	200	A	.	.	B	.	.	.	2.68	1.47	.	.	.	0.60	0.04
Phe	201	A	.	.	B	.	.	.	2.17	0.79	.	.	.	0.60	0.12
Val	202	A	.	.	B	.	.	.	2.09	0.53	.	.	.	0.60	0.16
Cys	203	A	.	.	.	.	T	.	2.04	0.71	.	.	.	0.20	0.17
Lys	204	A	.	.	.	.	T	.	1.74	0.76	.	.	.	0.20	0.17
Ser	205	A	.	.	.	.	T	.	0.84	0.89	.	.	.	0.20	0.24
Leu	206	A	.	.	.	.	T	.	0.10	0.24	.	.	.	0.10	0.88
Leu	207	A	A	.	.	.	.	.	0.10	0.33	.	.	.	0.30	0.88
Trp	208	A	A	.	.	.	.	.	0.24	0.31	.	.	.	0.30	0.49
Lys	209	A	A	.	.	.	.	.	0.50	0.61	.	.	.	0.60	0.49
Lys	210	A	A	.	.	.	.	.	0.44	0.36	*	.	.	0.30	0.91
Val	211	A	A	.	.	.	.	.	0.44	0.43	*	*	.	0.45	1.36
Leu	212	.	A	B	.	.	.	.	0.41	0.20	*	*	.	0.30	0.56
Pro	213	.	A	B	.	.	.	.	0.36	0.20	*	.	.	0.30	0.56
Tyr	214	.	.	.	B	T	.	.	0.58	0.63	*	.	.	0.20	0.75
Leu	215	.	.	.	B	T	.	.	1.29	0.67	*	*	.	0.20	0.64
Lys	216	.	.	.	B	T	.	.	0.73	0.56	*	.	.	0.20	0.22
Gly	217	.	.	B	B	.	.	.	0.27	0.51	*	.	.	0.60	0.19
Ile	218	.	.	B	B	.	.	.	0.40	0.19	*	.	.	0.30	0.23
Cys	219	.	.	B	.	.	T	.	0.50	0.07	*	.	.	0.70	0.11
Ser	220	.	.	.	.	T	T	.	0.03	0.36	.	*	F	0.65	0.11
Gly	221	.	.	.	.	T	T	.	0.08	0.36	.	.	F	0.65	0.16
Gly	222	.	.	.	.	T	T	.	0.06	0.33	.	.	F	1.25	0.49
Gly	223	.	.	.	.	.	.	C	0.94	0.47	.	.	F	0.85	0.57
Gly	224	.	.	.	.	.	.	C	1.72	0.86	*	.	F	1.15	0.99
Asp	225	.	.	.	.	.	T	C	1.17	1.29	.	*	F	1.50	1.97
Pro	226	.	.	.	.	.	T	C	1.51	1.07	*	.	F	1.84	1.47
Glu	227	.	.	B	.	.	T	.	1.97	1.50	*	.	F	1.98	2.49
Arg	228	.	.	B	.	.	T	.	2.01	1.93	*	.	F	2.32	2.92
Val	229	.	.	.	.	T	.	.	2.06	1.54	*	.	F	2.86	2.53
Asp	230	.	.	.	.	T	T	.	2.06	1.59	*	.	F	3.40	1.96
Arg	231	.	.	.	.	T	T	.	2.38	1.19	*	*	F	3.06	1.73
Ser	232	.	.	.	.	T	T	.	2.17	1.19	*	.	F	2.72	4.57
Ser	233	.	.	.	.	T	T	.	1.71	1.40	*	*	F	2.72	4.23
Gln	234	.	.	.	.	.	.	C	1.98	0.97	*	*	F	2.32	2.14
Arg	235	.	.	.	.	.	T	C	1.98	0.47	*	*	F	2.22	1.61
Pro	236	.	.	.	.	.	T	C	1.87	0.86	*	*	F	2.86	2.08
Gly	237	.	.	.	.	T	T	.	2.17	1.24	.	*	F	3.40	2.01
Ala	238	.	.	.	.	.	T	C	1.61	1.24	.	*	F	2.86	1.65
Glu	239	A	.	.	.	.	.	.	0.80	0.60	.	*	F	1.97	0.79
Asp	240	A	.	.	.	.	.	.	0.69	0.34	.	*	F	1.33	0.66
Asn	241	A	.	.	.	.	.	.	0.90	0.37	*	.	.	0.99	1.05
Val	242	A	.	.	.	.	.	.	0.36	0.87	*	.	.	0.95	1.05
Leu	243	A	.	.	.	.	.	.	0.09	0.19	*	.	.	0.50	0.44
Asn	244	A	.	.	B	.	.	.	0.21	0.46	*	.	.	0.60	0.20
Glu	245	A	.	.	B	.	.	.	1.10	0.44	*	.	.	0.60	0.37
Ile	246	A	.	.	B	.	.	.	1.91	0.49	*	.	.	0.60	0.31
Val	247	A	.	.	B	.	.	.	1.06	0.49	*	.	.	0.60	0.16
Ser	248	.	.	B	B	.	.	.	0.46	0.49	*	.	.	0.60	0.16
Ile	249	.	.	B	B	.	.	.	0.77	0.91	*	.	.	0.60	0.35
Leu	250	.	.	.	B	.	.	C	0.77	0.71	.	.	.	0.40	0.69
Gln	251	.	.	.	.	.	T	C	0.73	0.47	.	.	F	0.15	0.89
Pro	252	.	.	.	.	.	T	C	0.09	0.73	.	.	F	0.15	0.94
Thr	253	.	.	.	.	.	T	C	0.21	0.47	.	.	F	0.30	1.76
Gln	254	.	.	.	.	.	T	C	1.10	0.21	.	.	F	1.20	1.76
Val	255	.	A	.	.	.	.	C	1.91	0.21	.	.	F	0.80	1.97
Pro	256	.	A	.	.	.	.	C	1.31	0.64	.	.	F	1.10	2.37
Glu	257	A	A	.	.	.	.	.	1.52	0.51	.	*	F	0.90	1.35
Gln	258	A	A	.	.	.	.	.	0.98	0.91	.	*	F	0.90	3.16
Glu	259	A	A	.	.	.	.	.	0.98	0.91	.	*	F	0.90	1.51
Met	260	A	A	.	.	.	.	.	1.83	0.94	.	*	F	0.90	1.51
Glu	261	A	A	.	.	.	.	.	1.83	0.94	.	*	.	0.75	1.51
Val	262	A	A	.	.	.	.	.	1.24	0.91	.	*	F	0.90	1.35
Gln	263	A	A	.	.	.	.	.	1.24	0.41	.	*	F	0.60	1.38
Glu	264	A	A	.	.	.	.	.	1.03	1.03	.	*	F	0.90	1.38
Pro	265	A	A	.	.	.	.	.	1.32	0.60	.	*	F	1.18	2.88
Ala	266	A	A	.	.	.	.	.	0.98	0.76	.	*	F	1.46	2.40
Glu	267	A	.	.	.	.	T	.	0.98	0.73	.	*	F	2.14	1.37
Pro	268	A	.	.	.	.	T	.	0.98	0.09	.	.	F	1.97	0.66
Thr	269	.	.	.	.	T	T	.	0.38	0.11	.	.	F	2.80	1.05
Gly	270	A	.	.	.	.	T	.	0.22	0.00	.	.	F	1.37	0.60
Val	271	A	.	.	.	.	.	.	0.07	0.69	.	.	.	0.44	0.32
Asn	272	.	.	B	.	.	.	.	0.14	0.64	.	.	.	0.16	0.30
Met	273	.	.	B	.	.	.	.	0.28	0.59	.	.	.	0.18	0.46
Leu	274	.	.	.	.	.	.	C	0.03	0.59	.	.	.	0.40	0.62
Ser	275	.	.	.	.	.	T	C	0.08	0.06	.	.	F	1.95	0.66
Pro	276	.	.	.	.	.	T	C	0.93	0.07	.	.	F	2.25	0.90
Gly	277	.	.	.	.	.	T	C	0.90	0.69	.	.	F	3.00	1.89
Glu	278	A	.	.	.	.	T	.	0.69	0.87	.	.	F	2.50	1.92
Ser	279	A	A	.	.	.	.	.	0.69	0.57	.	.	F	1.80	1.02
Glu	280	A	A	.	.	.	.	.	0.99	0.31	.	.	F	1.05	0.85
His	281	A	A	.	.	.	.	.	0.99	0.74	.	.	F	1.05	0.85
Leu	282	A	A	.	.	.	.	.	0.74	0.31	.	.	.	0.30	0.98
Leu	283	A	A	.	.	.	.	.	0.74	0.20	.	.	.	0.30	0.57
Glu	284	A	A	.	.	.	.	.	0.46	0.20	.	.	F	0.45	0.73
Pro	285	A	A	.	.	.	.	.	0.46	0.20	.	.	F	0.45	0.89
Ala	286	A	A	.	.	.	.	.	0.60	0.89	.	.	F	0.90	1.88
Glu	287	A	A	.	.	.	.	.	1.11	1.57	.	.	F	0.90	2.13
Ala	288	A	A	.	.	.	.	.	1.92	1.19	.	.	F	0.90	1.84
Glu	289	A	A	.	.	.	.	.	2.03	1.21	*	.	F	0.90	3.16
Arg	290	A	A	.	.	.	.	.	2.36	1.71	*	.	F	0.90	3.57
Ser	291	A	.	.	.	.	T	.	3.06	1.71	*	.	F	1.30	6.92
Gln	292	A	.	.	.	.	T	.	2.24	2.21	*	.	F	1.30	7.83
Arg	293	A	.	.	.	.	T	.	2.02	1.53	.	.	F	1.30	3.30
Arg	294	A	.	.	.	.	T	.	1.17	0.84	.	.	F	1.30	2.03
Arg	295	.	.	.	B	T	.	.	0.84	0.59	.	*	F	1.15	0.87
Leu	296	.	.	B	B	.	.	.	0.56	0.56	.	*	.	0.60	0.69
Leu	297	.	.	B	B	.	.	.	0.56	0.06	.	*	.	0.30	0.35
Val	298	.	.	.	B	.	.	C	0.44	0.34	*	*	.	0.20	0.29
Pro	299	.	.	.	.	.	T	C	0.01	0.34	*	.	.	0.90	0.61
Ala	300	.	.	.	.	.	T	C	0.12	0.09	*	*	F	1.35	0.73
Asn	301	.	.	.	.	.	T	C	0.48	0.60	.	.	F	2.70	1.65
Glu	302	.	.	.	.	.	T	C	0.98	0.81	.	.	F	3.00	1.65
Gly	303	.	.	.	.	.	.	C	1.83	0.76	.	.	F	2.50	2.35
Asp	304	.	.	.	.	.	T	C	1.73	1.26	.	.	F	2.40	2.54
Pro	305	.	.	.	.	.	T	C	1.51	1.17	.	*	F	2.10	2.11
Thr	306	A	.	.	.	.	T	.	1.62	0.49	.	*	F	1.30	1.76
Glu	307	A	.	.	.	.	T	.	1.62	0.91	*	*	F	1.30	2.07
Thr	308	A	.	.	B	.	.	.	1.30	0.51	*	*	F	0.90	2.31
Leu	309	A	.	.	B	.	.	.	0.60	0.37	*	*	F	0.45	0.86
Arg	310	A	.	.	B	.	.	.	0.81	0.07	*	*	.	0.30	0.43
Gln	311	A	.	.	B	.	.	.	1.12	0.07	*	*	.	0.30	0.50
Cys	312	A	.	.	.	.	T	.	0.42	0.56	*	*	.	1.15	1.01
Phe	313	A	.	.	.	.	T	.	0.14	0.46	*	*	.	0.70	0.45
Asp	314	.	.	.	.	T	T	.	0.96	0.04	*	*	.	0.50	0.26
Asp	315	A	.	.	.	.	T	.	0.03	0.36	*	*	.	0.70	0.81
Phe	316	A	A	.	.	.	.	.	0.82	0.24	*	.	.	0.30	0.77
Ala	317	A	A	.	.	.	.	.	0.37	0.39	*	.	.	0.30	0.34
Asp	318	A	A	.	.	.	.	.	0.37	0.04	*	*	.	0.30	0.32
Leu	319	A	A	.	.	.	.	.	0.37	0.83	.	.	.	0.60	0.32
Val	320	.	A	.	.	.	.	C	0.67	0.04	.	.	.	0.10	0.52
Pro	321	.	A	.	.	.	.	C	0.26	0.07	.	.	.	0.50	0.42
Phe	322	.	.	.	.	T	T	.	0.33	0.84	.	.	.	0.20	0.54
Asp	323	A	.	.	.	.	T	.	0.12	0.16	.	.	.	0.25	1.25
Ser	324	A	.	.	.	.	T	.	0.12	0.06	.	.	F	1.00	1.25
Trp	325	A	.	.	.	.	T	.	0.38	0.20	*	*	F	0.40	1.19
Glu	326	A	A	.	.	.	.	.	0.70	0.03	*	.	F	0.15	0.71
Pro	327	A	A	.	.	.	.	.	1.44	0.03	*	.	.	0.15	1.03
Leu	328	A	A	.	.	.	.	.	0.63	0.36	*	.	.	0.45	1.96
Met	329	A	A	.	.	.	.	.	0.59	0.59	*	.	.	0.60	0.93
Arg	330	A	A	.	.	.	.	.	0.07	0.16	*	.	.	0.30	0.60
Lys	331	A	A	.	.	.	.	.	0.53	0.10	*	.	.	0.30	0.60
Leu	332	A	A	.	.	.	.	.	0.32	0.03	*	.	.	0.30	0.60
Gly	333	A	A	.	.	.	.	.	0.49	0.59	*	.	.	0.60	0.51
Leu	334	A	A	.	.	.	.	.	1.09	0.19	*	.	.	0.30	0.41
Met	335	A	A	.	.	.	.	.	0.09	0.19	*	*	.	0.30	0.86
Asp	336	A	A	.	.	.	.	.	0.09	0.19	.	*	F	0.45	0.61
Asn	337	A	A	.	.	.	.	.	0.04	0.61	*	*	F	0.90	1.48
Glu	338	A	A	.	.	.	.	.	0.20	0.66	*	*	F	0.90	1.11
Ile	339	A	A	.	.	.	.	.	0.66	0.77	*	*	F	0.75	0.67
Lys	340	A	A	.	.	.	.	.	0.67	0.77	.	*	F	0.75	0.83
Val	341	A	A	.	.	.	.	.	0.67	0.67	.	*	.	0.60	0.49
Ala	342	A	A	.	.	.	.	.	0.08	0.67	.	.	.	0.75	1.20
Lys	343	A	A	.	.	.	.	.	0.51	0.86	.	*	.	0.60	0.61
Ala	344	A	A	.	.	.	.	.	0.03	0.36	.	*	.	0.30	0.83
Glu	345	A	A	.	.	.	.	.	0.04	0.57	*	.	.	0.60	0.81
Ala	346	A	A	.	.	.	.	.	0.92	0.57	*	.	.	0.60	0.55
Ala	347	A	A	.	.	.	.	.	1.51	0.57	.	*	.	0.75	1.07
Gly	348	A	.	.	.	.	.	.	1.16	1.07	.	*	.	0.95	1.03
His	349	A	.	.	.	.	T	.	0.93	0.59	.	.	.	1.15	1.47
Arg	350	A	.	.	.	.	T	.	0.69	0.40	.	.	F	1.00	1.20
Asp	351	A	.	.	.	.	T	.	0.97	0.14	.	.	F	1.00	1.90
Thr	352	A	.	.	.	.	T	.	0.96	0.09	.	.	F	1.00	2.02
Leu	353	A	.	.	B	.	.	.	0.49	0.03	.	.	.	0.15	1.02
Tyr	354	A	.	.	B	.	.	.	0.37	0.71	.	*	.	0.60	0.50
Thr	355	A	.	.	B	.	.	.	0.43	1.40	.	*	.	0.60	0.24
Met	356	A	.	.	B	.	.	.	0.72	0.91	*	.	.	0.60	0.59
Leu	357	A	.	.	B	.	.	.	1.27	1.14	*	.	.	0.60	0.40
Ile	358	A	.	.	B	.	.	.	0.46	1.03	*	*	.	0.60	0.20
Lys	359	A	.	.	B	.	.	.	0.17	0.94	*	*	.	0.60	0.33
Trp	360	A	.	.	B	.	.	.	0.17	0.33	*	*	.	0.00	0.81
Val	361	A	.	.	B	.	.	.	0.09	0.13	*	*	.	0.45	1.66
Asn	362	.	.	.	.	.	T	C	1.01	0.13	*	.	F	1.95	0.82
Lys	363	.	.	.	.	.	T	C	1.90	0.13	*	*	F	2.40	1.53
Thr	364	.	.	.	.	.	T	C	1.27	1.04	*	.	F	3.00	3.44
Gly	365	.	.	.	.	.	T	C	1.26	1.19	*	.	F	2.70	2.16
Arg	366	.	A	.	.	T	.	.	1.26	1.20	*	.	F	2.20	1.45
Asp	367	.	A	.	.	.	.	C	1.22	0.56	*	.	F	1.55	0.75
Ala	368	A	A	.	.	.	.	.	0.87	0.54	.	.	F	1.20	1.03
Ser	369	A	A	.	.	.	.	.	0.37	0.49	.	.	.	0.30	0.76
Val	370	A	A	.	.	.	.	.	0.10	0.20	.	*	.	0.30	0.37
His	371	A	A	.	.	.	.	.	0.21	0.89	.	*	.	0.60	0.30
Thr	372	A	A	.	.	.	.	.	0.80	0.39	*	*	.	0.30	0.38
Leu	373	A	A	.	.	.	.	.	1.02	0.50	*	*	.	0.60	0.52
Leu	374	A	A	.	.	.	.	.	0.72	0.54	*	.	.	0.60	0.31
Asp	375	A	A	.	.	.	.	.	0.18	0.04	*	.	.	0.30	0.38
Ala	376	A	A	.	.	.	.	.	0.96	0.04	*	.	.	0.30	0.66
Leu	377	A	A	.	.	.	.	.	0.99	0.04	*	.	.	0.30	0.66
Glu	378	A	A	.	.	.	.	.	0.18	0.21	*	.	.	0.30	0.39
Thr	379	A	A	.	.	.	.	.	0.74	0.21	*	*	F	0.45	0.67
Leu	380	A	A	.	.	.	.	.	0.07	0.71	*	.	F	0.90	1.59
Gly	381	A	A	.	.	.	.	.	0.07	0.71	*	.	F	0.75	0.76
Glu	382	A	A	.	.	.	.	.	0.79	0.21	*	.	F	0.45	0.53
Arg	383	A	A	.	.	.	.	.	0.79	0.70	*	.	F	0.90	1.28
Leu	384	A	A	.	.	.	.	.	1.14	0.99	*	*	F	0.90	2.24
Ala	385	A	A	.	.	.	.	.	1.07	1.41	*	*	F	0.90	2.59
Lys	386	A	A	.	.	.	.	.	1.41	0.73	*	.	F	0.75	0.93
Gln	387	A	A	.	.	.	.	.	1.41	0.73	*	*	F	0.90	1.95
Lys	388	A	A	.	.	.	.	.	1.27	1.41	*	*	F	0.90	3.22
Ile	389	A	A	.	.	.	.	.	1.27	1.41	.	*	F	0.90	2.19
Glu	390	A	A	.	.	.	.	.	1.04	0.73	*	*	F	0.90	1.04
Asp	391	A	A	.	.	.	.	.	0.70	0.44	.	*	F	0.45	0.43
His	392	A	A	.	.	.	.	.	0.40	0.06	*	*	.	0.30	0.82
Leu	393	A	A	.	.	.	.	.	0.01	0.36	*	*	.	0.30	0.64
Leu	394	A	A	.	.	.	.	.	0.94	0.07	*	*	F	0.15	0.38
Ser	395	A	.	.	.	.	T	.	0.24	0.07	*	*	F	0.25	0.55
Ser	396	A	.	.	.	.	T	.	0.36	0.36	*	*	F	0.25	0.58
Gly	397	.	.	.	.	T	T	.	0.57	0.29	.	.	F	0.65	0.70
Lys	398	A	.	.	.	.	T	.	0.57	0.36	.	.	F	0.25	0.82
Phe	399	A	A	.	.	.	.	.	0.24	0.66	.	.	.	0.60	0.50
Met	400	.	A	B	.	.	.	.	0.20	0.27	.	*	.	0.30	0.88
Tyr	401	.	A	B	.	.	.	.	0.50	0.27	.	*	.	0.30	0.44
Leu	402	A	A	.	.	.	.	.	0.26	0.67	.	*	.	0.60	0.81
Glu	403	A	A	.	.	.	.	.	0.21	0.39	.	*	.	0.30	0.82
Gly	404	A	.	.	.	.	.	.	0.61	0.23	.	*	F	0.65	0.88
Asn	405	A	.	.	.	.	T	.	0.62	0.60	.	*	F	1.30	1.43
Ala	406	A	.	.	.	.	T	.	0.27	0.79	.	*	F	1.15	0.83
Asp	407	A	.	.	.	.	T	.	0.78	0.17	.	*	F	0.85	0.83
Ser	408	A	.	.	.	.	T	.	0.39	0.21	.	*	F	0.85	0.69
Ala	409	A	.	.	.	.	.	.	0.34	0.19	.	*	.	0.50	0.88
Met	410	A	.	.	.	.	.	.	0.04	0.26	.	.	.	0.50	0.67
Ser	411	A	.	.	.	.	.	.	0.16	0.17	.	.	.	0.10	0.64

[0096] In another aspect, the invention provides an antibody that binds a peptide or polypeptide comprising, or alternatively, consisting of, one, two, three, four, five or more, epitope-bearing portions of a TR7. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An immunogenic epitope is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an antigenic epitope. The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983).

[0097] As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, J. G. Sutcliffe et al., Antibodies That React With Predetermined Sites on Proteins, Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

[0098] Antigenic epitope-bearing peptides and polypeptides are therefore useful to raise antibodies, including monoclonal antibodies, that bind to a TR7 polypeptide. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and polypeptides preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of SEQ ID NO: 3.

[0099] Antibodies of the invention may bind one or more antigenic TR7 polypeptides or peptides including, but not limited to: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 62 to about 110 of SEQ ID NO: 3, about 119 to about 164 of SEQ ID NO: 3, about 224 to about 271 of SEQ ID NO: 3, about 275 to about 370 of SEQ ID NO: 3, about 69 to about 80 of SEQ ID NO: 3, about 88 to about 95 of SEQ ID NO: 3, about 99 to about 103 of SEQ ID NO: 3, about 119 to about 123 of SEQ ID NO: 3, about 130 to about 135 of SEQ ID NO: 3, about 152 to about 163 of SEQ ID NO: 3, about 226 to about 238 of SEQ ID NO: 3, about 275 to about 279 of SEQ ID NO: 3, about 301 to about 305 of SEQ ID NO: 3, and/or about 362 to about 367 of SEQ ID NO: 3. In this context about includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR7 receptor protein.

[0100] Epitope-bearing TR7 peptides and polypeptides may be produced by any conventional means. R. A. Houghten, General Method for the Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This Simultaneous Multiple Peptide Synthesis (SMPS) process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0101] As one of skill in the art will appreciate, TR7 receptor polypeptides and the epitope-bearing fragments thereof described herein (e.g., corresponding to a portion of the extracellular domain, such as, for example, amino acid residues 52 to 184 of SEQ ID NO: 3 can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR7 protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). TR7 fusion proteins may be used as an immunogen to elicit anti-TR7 antibodies. Thus, antibodies of the invention may bind the TR7 moirty of fusion proteins that comprise all or a portion of a TR7 polypeptide.

[0102] Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Antibodies of the present invention may also bind such modified TR7 polypeptides or TR7 polypeptide fragments or variants.

[0103] For instance, for many proteins, including the extracellular domain of a membrane associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function or loss of the ability to be bound by a specific antibody. However, even if deletion of one or more amino acids from the N-terminus or C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other TR7 functional activities may still be retained. For example, in many instances, the ability of the shortened protein to induce and/or bind to antibodies which recognize TR7 (preferably antibodies that bind specifically to TR7) will retained irrespective of the size or location of the deletion. In fact, polypeptides composed of as few as six TR7 amino acid residues may often evoke an immune response. Whether a particular polypeptide lacking N-terminal and/or C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.

[0104] As mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR7 ligand) may still be retained. For example, the ability of shortened TR7 polypeptides to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR7 polypeptide with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities.

[0105] Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the TR7 amino acid sequence shown in SEQ ID NO: 3 up to the alanine residue at position number 406 and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n⁵-411 of SEQ ID NO: 3 where n⁵ is an integer from 2 to 406 corresponding to the position of the amino acid residue in SEQ ID NO: 3.

[0106] More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: E-2 to S-411; Q-3 to S-411; R-4 to S-411; G-5 to S-411; Q-6 to S-411; N-7 to S-411; A-8 to S-411; P-9 to S-411; A-10 to S-411; A-11 to S-411; S-12 to S-411; G-13 to S-411; A-14 to S-411; R-15 to S-411; K-16 to S-411; R-17 to S-411; H-18 to S-411; G-19 to S-411; P-20 to S-411; G-21 to S-411; P-22 to S-411; R-23 to S-411; E-24 to S-411; A-25 to S-411; R-26 to S-411; G-27 to S-411; A-28 to S-411; R-29 to S-411; P-30 to S-411; G-31 to S-411; G-31 to S-411; P-32 to S-411; R-33 to S-411; V-34 to S-411; P-35 to S-411; K-36 to S-411; T-37 to S-411; L-38 to S-411; V-39 to S-411; L-40 to S-411; V-41 to S-411; V-42 to S-411; A-43 to S-411; A-44 to S-411; V-45 to S-411; L-46 to S-411; L-47 to S-411; L-48 to S-411; V-49 to S-411; S-50 to S-411; A-51 to S-411; E-52 to S-411; S-53 to S-411; A-54 to S-411; L-55 to S-411; I-56 to S-411; T-57 to S-411; Q-58 to S-411; Q-59 to S-411; D-60 to S-411; L-61 to S-411; A-62 to S-411; P-63 to S-411; Q-64 to S-411; Q-65 to S-411; R-66 to S-411; A-67 to S-411; A-68 to S-411; P-69 to S-411; Q-70 to S-411; Q-71 to S-411; K-72 to S-411; R-73 to S-411; S-74 to S-411; S-75 to S-411; P-76 to S-411; S-77 to S-411; E-78 to S-411; G-79 to S-411; L-80 to S-411; C-81 to S-411; P-82 to S-411; P-83 to S-411; G-84 to S-411; H-85 to S-411; H-86 to S-411; I-87 to S-411; S-88 to S-411; E-89 to S-411; D-90 to S-411; G-91 to S-411; R-92 to S-411; D-93 to S-411; C-94 to S-411; I-95 to S-411; S-96 to S-411; C-97 to S-411; K-98 to S-411; Y-99 to S-411; G-100 to S-411; Q-101 to S-411; D-102 to S-411; Y-103 to S-411; S-104 to S-411; T-105 to S-411; H-106 to S-411; W-107 to S-411; N-108 to S-411; D-109 to S-411; L-110 to S-411; L-111 to S-411; F-112 to S-411; C-113 to S-411; L-114 to S-411; R-115 to S-411; C-116 to S-411; T-117 to S-411; R-118 to S-411; C-119 to S-411; D-120 to S-411; S-121 to S-411; G-122 to S-411; E-123 to S-411; V-124 to S-411; E-125 to S-411; L-126 to S-411; S-127 to S-411; P-128 to S-411; C-129 to S-411; T-130 to S-411; T-131 to S-411; T-132 to S-411; R-133 to S-411; N-134 to S-411; T-135 to S-411; V-136 to S-411; C-137 to S-411; Q-138 to S-411; C-139 to S-411; E-140 to S-411; E-141 to S-411; G-142 to S-411; T-143 to S-411; F-144 to S-411; R-145 to S-411; E-146 to S-411; E-147 to S-411; D-148 to S-411; S-149 to S-411; P-150 to S-411; E-151 to S-411; M-152 to S-411; C-153 to S-411; R-154 to S-411; K-155 to S-411; C-156 to S-411; R-157 to S-411; T-158 to S-411; G-159 to S-411; C-160 to S-411; P-161 to S-411; R-162 to S-411; G-163 to S-411; M-164 to S-411; V-165 to S-411; K-166 to S-411; V-167 to S-411; G-168 to S-411; D-169 to S-411; C-170 to S-411; T-171 to S-411; P-172 to S-411; W-173 to S-411; S-174 to S-411; D-175 to S-411; I-176 to S-411; E-177 to S-411; C-178 to S-411; V-179 to S-411; H-180 to S-411; K-181 to S-411; E-182 to S-411; S-183 to S-411; G-184 to S-411; I-185 to S-411; I-186 to S-411; I-187 to S-411; G-188 to S-411; V-189 to S-411; T-190 to S-411; V-191 to S-411; A-192 to S-411; A-193 to S-411; V-194 to S-411; V-195 to S-411; L-196 to S-411; I-197 to S-411; V-198 to S-411; A-199 to S-411; V-200 to S-411; F-201 to S-411; V-202 to S-411; C-203 to S-411; K-204 to S-411; S-205 to S-411; L-206 to S-411; L-207 to S-411; W-208 to S-411; K-209 to S-411; K-210 to S-411; V-211 to S-411; L-212 to S-411; P-213 to S-411; Y-214 to S-411; L-215 to S-411; K-216 to S-411; G-217 to S-411; I-218 to S-411; C-219 to S-411; S-220 to S-411; G-221 to S-411; G-222 to S-411; G-223 to S-411; G-224 to S-411; D-225 to S-411; P-226 to S-411; E-227 to S-411; R-228 to S-411; V-229 to S-411; D-230 to S-411; R-231 to S-411; S-232 to S-411; S-233 to S-411; Q-234 to S-411; R-235 to S-411; P-236 to S-411; G-237 to S-411; A-238 to S-411; E-239 to S-411; D-240 to S-411; N-241 to S-411; V-242 to S-411; L-243 to S-411; N-244 to S-411; E-245 to S-411; I-246 to S-411; V-247 to S-411; S-248 to S-411; I-249 to S-411; L-250 to S-411; Q-251 to S-411; P-252 to S-411; T-253 to S-411; Q-254 to S-411; V-255 to S-411; P-256 to S-411; E-257 to S-411; Q-258 to S-411; E-259 to S-411; M-260 to S-411; E-261 to S-411; V-262 to S-411; Q-263 to S-411; E-264 to S-411; P-265 to S-411; A-266 to S-411; E-267 to S-411; P-268 to S-411; T-269 to S-411; G-270 to S-411; V-271 to S-411; N-272 to S-411; M-273 to S-411; L-274 to S-411; S-275 to S-411; P-276 to S-411; G-277 to S-411; E-278 to S-411; S-279 to S-411; E-280 to S-411; H-281 to S-411; L-282 to S-411; L-283 to S-411; E-284 to S-411; P-285 to S-411; A-286 to S-411; E-287 to S-411; A-288 to S-411; E-289 to S-411; R-290 to S-411; S-291 to S-411; Q-292 to S-411; R-293 to S-411; R-294 to S-411; R-295 to S-411; L-296 to S-411; L-297 to S-411; V-298 to S-411; P-299 to S-411; A-300 to S-411; N-301 to S-411; E-302 to S-411; G-303 to S-411; D-304 to S-411; P-305 to S-411; T-306 to S-411; E-307 to S-411; T-308 to S-411; L-309 to S-411; R-310 to S-411; Q-311 to S-411; C-312 to S-411; F-313 to S-411; D-314 to S-411; D-315 to S-411; F-316 to S-411; A-317 to S-411; D-318 to S-411; L-319 to S-411; V-320 to S-411; P-321 to S-411; F-322 to S-411; D-323 to S-411; S-324 to S-411; W-325 to S-411; E-326 to S-411; P-327 to S-411; L-328 to S-411; M-329 to S-411; R-330 to S-411; K-331 to S-411; L-332 to S-411; G-333 to S-411; L-334 to S-411; M-335 to S-411; D-336 to S-411; N-337 to S-411; E-338 to S-411; I-339 to S-411; K-340 to S-411; V-341 to S-411; A-342 to S-411; K-343 to S-411; A-344 to S-411; E-345 to S-411; A-346 to S-411; A-347 to S-411; G-348 to S-411; H-349 to S-411; R-350 to S-411; D-351 to S-411; T-352 to S-411; L-353 to S-411; Y-354 to S-411; T-355 to S-411; M-356 to S-411; L-357 to S-411; I-358 to S-411; K-359 to S-411; W-360 to S-411; V-361 to S-411; N-362 to S-411; K-363 to S-411; T-364 to S-411; G-365 to S-411; R-366 to S-411; D-367 to S-411; A-368 to S-411; S-369 to S-411; V-370 to S-411; H-371 to S-411; T-372 to S-411; L-373 to S-411; L-374 to S-411; D-375 to S-411; A-376 to S-411; L-377 to S-411; E-378 to S-411; T-379 to S-411; L-380 to S-411; G-381 to S-411; E-382 to S-411; R-383 to S-411; L-384 to S-411; A-385 to S-411; K-386 to S-411; Q-387 to S-411; K-388 to S-411; I-389 to S-411; E-390 to S-411; D-391 to S-411; H-392 to S-411; L-393 to S-411; L-394 to S-411; S-395 to S-411; S-396 to S-411; G-397 to S-411; K-398 to S-411; F-399 to S-411; M-400 to S-411; Y-401 to S-411; L-402 to S-411; E-403 to S-411; G-404 to S-411; N-405 to S-411; and/or A-406 to S-411 of the TR7 sequence shown in SEQ ID NO: 3.

[0107] In another embodiment, N-terminal deletions of the TR7 polypeptide can be described by the general formula n⁶ to 184 where n⁶ is a number from 1 to 179 corresponding to the amino acid sequence identified in SEQ ID NO: 3. In specific embodiments, antibodies of the invention bind N terminal deletions of the TR7 comprising, or alternatively consisting of, the amino acid sequence of residues: E-2 to G-184; Q-3 to G-184; R-4 to G-184; G-5 to G-184; Q-6 to G-184; N-7 to G-184; A-8 to G-184; P-9 to G-184; A-10 to G-184; A-11 to G-184; S-12 to G-184; G-13 to G-184; A-14 to G-184; R-15 to G-184; K-16 to G-184; R-17 to G-184; H-18 to G-184; G-19 to G-184; P-20 to G-184; G-21 to G-184; P-22 to G-184; R-23 to G-184; E-24 to G-184; A-25 to G-184; R-26 to G-184; G-27 to G-184; A-28 to G-184; R-29 to G-184; P-30 to G-184; G-31 to G-184; P-32 to G-184; R-33 to G-184; V-34 to G-184; P-35 to G-184; K-36 to G-184; T-37 to G-184; L-38 to G-184; V-39 to G-184; L-40 to G-184; V-41 to G-184; V-42 to G-184; A-43 to G-184; A-44 to G-184; V-45 to G-184; L-46 to G-184; L-47 to G-184; L-48 to G-184; V-49 to G-184; S-50 to G-184; A-51 to G-184; E-52 to G-184; S-53 to G-184; A-54 to G-184; L-55 to G-184; I-56 to G-184; T-57 to G-184; Q-58 to G-184; Q-59 to G-184; D-60 to G-184; L-61 to G-184; A-62 to G-184; P-63 to G-184; Q-64 to G-184; Q-65 to G-184; R-66 to G-184; A-67 to G-184; A-68 to G-184; P-69 to G-184; Q-70 to G-184; Q-71 to G-184; K-72 to G-184; R-73 to G-184; S-74 to G-184; S-75 to G-184; P-76 to G-184; S-77 to G-184; E-78 to G-184; G-79 to G-184; L-80 to G-184; C-81 to G-184; P-82 to G-184; P-83 to G-184; G-84 to G-184; H-85 to G-184; H-86 to G-184; I-87 to G-184; S-88 to G-184; E-89 to G-184; D-90 to G-184; G-91 to G-184; R-92 to G-184; D-93 to G-184; C-94 to G-184; I-95 to G-184; S-96 to G-184; C-97 to G-184; K-98 to G-184; Y-99 to G-184; G-100 to G-184; Q-101 to G-184; D-102 to G-184; Y-103 to G-184; S-104 to G-184; T-105 to G-184; H-106 to G-184; W-107 to G-184; N-108 to G-184; D-109 to G-184; L-110 to G-184; L-111 to G-184; F-112 to G-184; C-113 to G-184; L-114 to G-184; R-115 to G-184; C-116 to G-184; T-117 to G-184; R-118 to G-184; C-119 to G-184; D-120 to G-184; S-121 to G-184; G-122 to G-184; E-123 to G-184; V-124 to G-184; E-125 to G-184; L-126 to G-184; S-127 to G-184; P-128 to G-184; C-129 to G-184; T-130 to G-184; T-131 to G-184; T-132 to G-184; R-133 to G-184; N-134 to G-184; T-135 to G-184; V-136 to G-184; C-137 to G-184; Q-138 to G-184; C-139 to G-184; E-140 to G-184; E-141 to G-184; G-142 to G-184; T-143 to G-184; F-144 to G-184; R-145 to G-184; E-146 to G-184; E-147 to G-184; D-148 to G-184; S-149 to G-184; P-150 to G-184; E-151 to G-184; M-152 to G-184; C-153 to G-184; R-154 to G-184; K-155 to G-184; C-156 to G-184; R-157 to G-184; T-158 to G-184; G-159 to G-184; C-160 to G-184; P-161 to G-184; R-162 to G-184; G-163 to G-184; M-164 to G-184; V-165 to G-184; K-166 to G-184; V-167 to G-184; G-168 to G-184; D-169 to G-184; C-170 to G-184; T-171 to G-184; P-172 to G-184; W-173 to G-184; S-174 to G-184; D-175 to G-184; I-176 to G-184; E-177 to G-184; C-178 to G-184; and/or V-179 to G-184; of the TR7 extracellular domain sequence shown in SEQ ID NO: 3.

[0108] Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR7 ligand (e.g., TRAIL)) may still be retained. For example, the ability of the shortened TR7 polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR7 polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR7 amino acid residues may often evoke an immune response.

[0109] Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR7 polypeptide shown in SEQ ID NO: 3 up to the glutamic acid residue at position number 52. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 52-m⁵ of SEQ ID NO: 3, where m⁵is an integer from 57 to 410 corresponding to the position of the amino acid residue in SEQ ID NO: 3.

[0110] More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: E-52 to M-410; E-52 to A-409; E-52 to S-408; E-52 to D-407; E-52 to A-406; E-52 to N-405; E-52 to G-404; E-52 to E-403; E-52 to L-402; E-52 to Y-401; E-52 to M-400; E-52 to F-399; E-52 to K-398; E-52 to G-397; E-52 to S-396; E-52 to S-395; E-52 to L-394; E-52 to L-393; E-52 to H-392; E-52 to D-391; E-52 to E-390; E-52 to I-389; E-52 to K-388; E-52 to Q-387; E-52 to K-386; E-52 to A-385; E-52 to L-384; E-52 to R-383; E-52 to E-382; E-52 to G-381; E-52 to L-380; E-52 to T-379; E-52 to E-378; E-52 to L-377; E-52 to A-376; E-52 to D-375; E-52 to L-374; E-52 to L-373; E-52 to T-372; E-52 to H-371; E-52 to V-370; E-52 to S-369; E-52 to A-368; E-52 to D-367; E-52 to R-366; E-52 to G-365; E-52 to T-364; E-52 to K-363; E-52 to N-362; E-52 to V-361; E-52 to W-360; E-52 to K-359; E-52 to I-358; E-52 to L-357; E-52 to M-356; E-52 to T-355; E-52 to Y-354; E-52 to L-353; E-52 to T-352; E-52 to D-351; E-52 to R-350; E-52 to H-349; E-52 to G-348; E-52 to A-347; E-52 to A-346; E-52 to E-345; E-52 to A-344; E-52 to K-343; E-52 to A-342; E-52 to V-341; E-52 to K-340; E-52 to I-339; E-52 to E-338; E-52 to N-337; E-52 to D-336; E-52 to M-335; E-52 to L-334; E-52 to G-333; E-52 to L-332; E-52 to K-331; E-52 to R-330; E-52 to M-329; E-52 to L-328; E-52 to P-327; E-52 to E-326; E-52 to W-325; E-52 to S-324; E-52 to D-323; E-52 to F-322; E-52 to P-321; E-52 to V-320; E-52 to L-319; E52 to D-318; E-52 to A-317; E-52 to F-316; E-52 to D-315; E-52 to D-314; E-52 to F-313; E-52 to C-312; E-52 to Q-311; E-52 to R-310; E-52 to L-309; E-52 to T-308; E-52 to E-307; E-52 to T-306; E-52 to P-305; E-52 to D-304; E-52 to G-303; E-52 to E-302; E-52 to N-301; E-52 to A-300; E-52 to P-299; E-52 to V-298; E-52 to L-297; E-52 to L-296; E-52 to R-295; E-52 to R-294; E-52 to R-293; E-52 to Q-292; E-52 to S-291; E-52 to R-290; E-52 to E-289; E-52 to A-288; E-52 to E-287; E-52 to A-286; E-52 to P-285; E-52 to E-284; E-52 to L-283; E-52 to L-282; E-52 to H-281; E-52 to E-280; E-52 to S-279; E-52 to E-278; E-52 to G-277; E-52 to P-276; E-52 to S-275; E-52 to L-274; E-52 to M-273; E-52 to N-272; E-52 to V-271; E-52 to G-270; E-52 to T-269; E-52 to P-268; E-52 to E-267; E-52 to A-266; E-52 to P-265; E-52 to E-264; E-52 to Q-263; E-52 to V-262; E-52 to E-261; E-52 to M-260; E-52 to E-259; E-52 to Q-258; E-52 to E-257; E-52 to P-256; E-52 to V-255; E-52 to Q-254; E-52 to T-253; E-52 to P-252; E-52 to Q-251; E-52 to L-250; E-52 to I-249; E-52 to S-248; E-52 to V-247; E-52 to I-246; E-52 to E-245; E-52 to N-244; E-52 to L-243; E-52 to V-242; E-52 to N-241; E-52 to D-240; E-52 to E-239; E-52 to A-238; E-52 to G-237; E-52 to P-236; E-52 to R-235; E-52 to Q-234; E-52 to S-233; E-52 to S-232; E-52 to R-231; E-52 to D-230; E-52 to V-229; E-52 to R-228; E-52 to E-227; E-52 to P-226; E-52 to D-225; E-52 to G-224; E-52 to G-223; E-52 to G-222; E-52 to G-221; E-52 to S-220; E-52 to C-219; E-52 to I-218; E-52 to G-217; E-52 to K-216; E-52 to L-215; E-52 to Y-214; E-52 to P-213; E-52 to L-212; E-52 to V-211; E-52 to K-210; E-52 to K-209; E-52 to W-208; E-52 to L-207; E-52 to L-206; E-52 to S-205; E-52 to K-204; E-52 to C-203; E-52 to V-202; E-52 to F-201; E-52 to V-200; E-52 to A-199; E-52 to V-198; E-52 to I-197; E-52 to L-196; E-52 to V-195; E-52 to V-194; E-52 to A-193; E-52 to A-192; E-52 to V-191; E-52 to T-190; E-52 to V-189; E-52 to G-188; E-52 to I-187; E-52 to I-186; E-52 to I-185; E-52 to G-184; E-52 to S-183; E-52 to E-182; E-52 to K-181; E-52 to H-180 E-52 to V-179; E-52 to C-178; E-52 to E-177; E-52 to I-176; E-52 to D-175; E-52 to S-174; E-52 to W-173; E-52 to P-172; E-52 to T-171; E-52 to C-170; E-52 to D-169; E-52 to G-168; E-52 to V-167; E-52 to K-166; E-52 to V-165; E-52 to M-164; E-52 to G-163; E-52 to R-162; E-52 to P-161; E-52 to C-160; E-52 to G-159; E-52 to T-158; E-52 to R-157; E-52 to C-156; E-52 to K-155; E-52 to R-154; E-52 to C-153; E-52 to M-152; E-52 to E-151; E-52 to P-150; E-52 to S-149; E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52 to R-145; E-52 to F-144; E-52 to T-143; E-52 to G-142; E-52 to E-141; E-52 to E-140; E-52 to C-139; E-52 to Q-138; E-52 to C-137; E-52 to V-136; E-52 to T-135; E-52 to N-134; E-52 to R-133; E-52 to T-132; E-52 to T-131; E-52 to T-130; E-52 to C-129; E-52 to P-128; E-52 to S-127; E-52 to L-126; E-52 to E-125; E-52 to V-124; E-52 to E-123; E-52 to G-122; E-52 to S-121; E-52 to D-120; E-52 to C-119; E-52 to R-118; E-52 to T-117; E-52 to C-116; E-52 to R-115; E-52 to L-114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-52 to L-110; E-52 to D-109; E-52 to N-108; E-52 to W-107; E-52 to H-106; E-52 to T-105; E-52 to S-104; E-52 to Y-103; E-52 to D-102; E-52 to Q-101; E-52 to G-100; E-52 to Y-99; E-52 to K-98; E-52 to C-97; E-52 to S-96; E-52 to I-95; E-52 to C-94; E-52 to D-93; E-52 to R-92; E-52 to G-91; E-52 to D-90; E-52 to E-89; E-52 to S-88; E-52 to I-87; E-52 I-87; E-52 to H-86; E-52 to H-85; E-52 to G-84; E-52 to P-83; E-52 to P-82; E-52 to C-81; E-52 to L-80; E-52 to G-79; E-52 to E-78; E-52 to S-77; E-52 to P-76; E-52 to S-75; E-52 to S-74; E-52 to R-73; E-52 to K-72; E-52 to Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68; E-52 to A-67; E-52 to R-66; E-52 to Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-62; E-52 to L-61; E-52 to D-60; E-52 to Q-59; E-52 to Q-58; and/or E-52 to T-57; of the TR7 sequence shown in SEQ ID NO: 3.

[0111] In another embodiment, antibodies of the invention bind C-terminal deletions of the TR7 polypeptide that can be described by the general formula 52-m⁶ where m⁶ is a number from 57 to 183 corresponding to the amino acid sequence identified in SEQ ID NO: 3. In specific embodiments, antibodies of the invention bind C terminal deletions of the TR7 polypeptide comprising, or alternatively, consisting of, amino acid residues: E-52 to S-183; E-52 to E-182; E-52 to K-181; E-52 to H-180; E-52 to V-179; E-52 to C-178; E-52 to E-177; E-52 to I-176; E-52 to D-175; E-52 to S-174; E-52 to W-173; E-52 to P-172; E-52 to T-171; E-52 to C-170; E-52 to D-169; E-52 to G-168; E-52 to V-167; E-52 to K-166; E-52 to V-165; E-52 to M-164; E-52 to G-163; E-52 to R-162; E-52 to P-161; E-52 to C-160; E-52 to G-159; E-52 to T-158; E-52 to R-157; E-52 to C-156; E-52 to K-155; E-52 to R-154; E-52to C-153; E-52 to M-152; E-52 to E-151; E-52 to P-150; E-52 to S-149; E-52 to D-148; E-52 to E-147; E-52 to E-146; E-52 to R-145; E-52 to F-144; E-52 to T-143; E-52 to G-142; E-52 to E-141; E-52 to E-140; E-52 to C-139; E-52 to Q-138; E-52 to C-137; E-52 to V-136; E-52 to T-135; E-52 to N-134; E-52 to R-133; E-52 to T-132; E-52 to T-131; E-52 to T-130; E-52 to C-129; E-52 to P-128; E-52 to S-127; E-52 to L-126; E-52 to E-125; E-52 to V-124; E-52 to E-123; E-52 to G-122; E-52 to S-121; E-52 to D-120; E-52 to C-119; E-52 to R-118; E-52 to T-117; E-52 to C-116; E-52 to R-115; E-52 to L-114; E-52 to C-113; E-52 to F-112; E-52 to L-111; E-52 to L-110; E-52 to D-109; E-52 to N-108; E-52 to W-107; E-52 to H-106; E-52 to T-105; E-52 to S-104; E-52 to Y-103; E-52 to D-102; E-52 to Q-101; E-52 to G-100; E-52 to Y-99; E-52 to K-98; E-52 to C-97; E-52 to S-96; E-52 to I-95; E-52 to C-94; E-52 to D-93; E-52 to R-92; E-52 to G-91; E-52 to D-90; E-52 to E-89; E-52 to S-88; E-52 to I-87; E-52 to H-86; E-52 to H-85; E-52 to G-84; E-52 to P-83; E-52 to P-82; E-52 to C-81; E-52 to L-80; E-52 to G-79; E-52; E-78; E-52 to S-77; E-52 to P-76; E-52 to S-75; E-52 to S-74; E-52 to R-73; E-52 to K-72; E-52 to Q-71; E-52 to Q-70; E-52 to P-69; E-52 to A-68; E-52 to A-67; E-52 to R-66; E-52 to Q-65; E-52 to Q-64; E-52 to P-63; E-52 to A-62; E-52 to L-61; E-52 to D-60; E-52 Q-59; E-52 to Q-58; and/or E-52 to T-57; of the TR7 extracellular domain sequence shown in SEQ ID NO: 3.

[0112] The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR7 polypeptide, which may be described generally as having residues n⁵m⁵ and/or n⁶m⁶ of SEQ ID NO: 3, where n⁵, n⁶, m⁵, and m⁶ are integers as described above.

[0113] Also included are antibodies that bind a polypeptide consisting of a portion of the complete TR7 amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97920, where this portion excludes from 1 to about 78 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97920, or from 1 to about 233 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97920.

[0114] Preferably, antibodies of the present invention bind the N- and C-terminal deletion mutants comprising only a portion of the extracellular domain; i.e., within residues 52-184,of SEQ ID NO: 3, since any portion therein is expected to be soluble.

[0115] It will be recognized in the art that some amino acid sequence of TR7 can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Such areas will usually comprise residues which make up the ligand binding site or the death domain, or which form tertiary structures which affect these domains.

[0116] Thus, the invention further includes antibodies that bind variations of the TR7 protein which show substantial TR7 protein activity or which include regions of TR7, such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U. et al., Science 247:1306-1310 (1990).

[0117] Thus, antibodies of the present invention may bind a fragment, derivative, or analog of the polypeptide of SEQ ID NO: 3, or that encoded by the cDNA in ATCC deposit 97920. Such fragments, variants or derivatives may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0118] Of particular interest are substitutions of charged amino acids with another charged amino acids and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TR7 protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical- formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

[0119] The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Thus, the antibodies of the present invention may bind a TR7 receptor that contains one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

[0120] As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 3).

TABLE 3
Conservative Amino Acid Substitutions.
	Aromatic	Phenylalanine
		Tryptophan
		Tyrosine
	Hydrophobic	Leucine
		Isoleucine
		Valine
	Polar	Glutamine
		Asparagine
	Basic	Arginine
		Lysine
		Histidine
	Acidic	Aspartic Acid
		Glutamic Acid
	Small	Alanine
		Serine
		Threonine
		Methionine
		Glycine